Synthetic fatty acid desaturase gene for expression in plants

ABSTRACT

A synthetic fatty acid desaturase gene for expression in a multicellular plant is provided. The gene comprises a desaturase domain and a cyt b 5  domain, and is customized for expression in a plant cytoplasm. Methods for designing and making a synthetic fatty acid desaturase gene customized for expression in a plant cytoplasm are also provided.

This application claims priority to U.S. Provisional Application No. 60/097,586, filed Aug. 24, 1998, the entirety of which is incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to the field of genetic engineering, and more particularly to transformation of plants with heterologous fatty acid desaturase genes modified for optimum expression in plants.

BACKGROUND OF THE INVENTION

Several publications are referenced in this application in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.

Alteration of fatty acid desaturation in plants is of interest to plant biologists and food scientists alike, due to the influence of unsaturated fatty acids on the health benefits and flavors of foods, as well as the role of these molecules in plant biological processes. For a nation interested in healthy diet, the quality of fats and oils depends on their fatty acid composition, with oils high in monounsaturated fatty acids (e.g., canola, olive) gaining popularity as new health benefits are discovered. Considering the flavors of plant foods, many flavor-producing compounds are derived from peroxidation of unsaturated fatty acids. Thus, efforts are being made to produce plants with increased amounts of unsaturated fatty acids, preferably monounsaturated fatty acids.

In animal and fungal cells, monounsaturated fatty acids are aerobically synthesized from saturated fatty acids by a microsomal Δ-9 fatty acid desaturase that is membrane bound and cytochrome b₅-dependent. A double bond is inserted between the 9- and 10-carbons of palmitoyl (16:0) and stearoyl (18:0) CoA to form palmitoleic (16:1) and oleic (18:1) acids. In the reaction mechanism, electrons are transferred from NADH-dependent cytochrome b₅ reductase, via the heme-containing cytochrome b₅ (Cyt b₅) molecule, to the Δ-9 fatty acid desaturase. The major form of cytochrome b₅ in animal, fungal and plant cells exists as an independent protein molecule that is anchored to the membrane by a short, carboxyl terminal, hydrophobic stretch of amino acids. The carboxyl terminal anchor orients the heme group of the Cyt b₅ on the membrane surface and allows it to translationally diffuse across the surface of the membrane. This property of lateral mobility allows this form of cytochrome b₅ to participate as an electron donor to a number of different proteins that catalyze a variety metabolic reactions on the membrane surface, including fatty acid desaturases, various sterol biosynthetic enzymes and a variety of cytochrome P450 mediated reactions. While this contributes to the versatility of Cyt b₅ as an electron donor, it also implies that the major form of cytochrome b₅ shuttles between its redox partners by translational diffusion across the surface of the membrane (Strittmatter and Rogers, Proc. Natl. Acad. Sci. USA, 72: 2658-2661, (1975; Lederer, Biochimie 76: 674-692, 1994). Furthermore, this mechanism suggests that an independent, membrane bound cytochrome b₅ molecule can potentially limit the rate of the metabolic reaction, depending on its abundance, its location on the membrane surface, its proximity to the electron acceptor, and the rate at which it can move and orient itself to the acceptor on the membrane surface.

In plants, unsaturated fatty acids are formed and incorporated into complex lipids in two distinct cellular compartments. De novo fatty acid synthesis occurs almost exclusively in the plastids, producing the saturated species 16:0-ACP (acyl carrier protein) and 18:0-ACP. 18:1-ACP is formed from 18:0-ACP in the plastid by a soluble, ferredoxin-dependent Δ-9 desaturase. These fatty acids are then shunted into one of two routes—a plastid-localized “procaryotic” pathway or a cytosolic/ER (endoplasmic reticulum) “eucaryotic” pathway—for further modification and acylation into glycerolipids (Somerville and Browse, Science 252: 80-87, 1991). The acyl ACPs that are shunted into the prokaryotic pathway remain within the plastid and are used for the synthesis of phosphatidic acid and further conversion to chloroplast glycerolipids. The fatty acyl groups of those lipids may be further desaturated by plastid desaturases that also use ferrodoxin as the electron donor.

Acyl-ACPs that are shunted into the eukaryotic pathway are converted to free fatty acids, transported across the chloroplast membrane into the cytoplasm where they are converted to acyl CoA thioesters by acyl CoA synthetase. Those fatty acids are then converted to cytoplasmic/ER phosphatidic acid which can then be converted to membrane glycerophospholipids, or storage lipids, in the form of triacylglycerols and sterol esters that are the major components of plant oils.

Most polyunsaturated 18-carbon plant fatty acids appear to be formed in the cytosol by the ER-bound desaturases (Table 1). Once the 18:1 fatty acid is incorporated into phospholipid, an ER-bound desaturase can catalyze the formation of a Δ-12 double bond in the fatty acyl chain to form Δ-9,12 18:2. Other ER bound desaturase enzymes can act on 18:2 to introduce a Δ-15 double bond to form Δ9,12,15 18:3. These desaturase are thought to be similar to animal and fungal desaturases because they are membrane bound and appear to require a cytochrome b₅-mediated electron transport chain.

TABLE 1 Desaturase Primary b5 Plant Gene Type Activity chimera Reference Arabidopsis FAD2 Δ12, 18:1->18:2 no Okuley J. et al. microsomal Plant Cell 6: 147-158, 1994 Arabidopsis FAD3 Δ15, 18:2->18:3 no Shah S. & Z. Xin, microsomal Plant Physiol. 114: 1533-1539, 1997 Nicotiana NtFA Δ15, 18:2->18:3 no Hamada T. et al. tabacum D3 microsomal Plant & Cell. Physiol. 37: 606-611, 1996, Hamada T. et al. Transgenic Res. 5: 115-121, 1996 Soybean FAD Δ12, 18:1-> no Heppard E. P. et al. 2-1 microsomal, 18:2 Plant Physiol. 110: developing 311-319, 1996 seeds Soybean FAD Δ12, 18:1->18:2 no Heppard, E. P. et al. 2-2 microsomal 1996, supra developing seeds and vegetative tissues Borage Δ-6 18:2 yes, N- Sayanova et al. (9, 12)-18:3 terminal Proc. Natl. Acad. (6, 9, 12) Sci. USA 94: 4211-4216, 1997

The conversion of saturated fatty acyl chains to monounsaturated species in plants appears to be confined to the chloroplasts. No Δ-9 desaturase activity has been identified in the cytoplasm or endoplasmic reticulum of plants. The soluble plant chloroplast Δ-9 desaturase is highly specific for 18:0-ACP as a substrate and does not desaturate 16:0-ACP (Somerville and Browse, supra). As a result, only a small amount of 16:1 is present in most higher plants, while the pool of 16:0 is concomitantly larger due to its disfavor as a substrate for the plant desaturase. By comparison, a larger amount of 18:1 is found in higher plant cells, with a correspondingly lesser amount of 18:0. Thus, for the purpose of increasing the concentration of mono-unsaturated lipids in a plant, the 16:0 fatty acid constitutes a significant pool of available substrate that is under-utilized by the endogenous plant desaturase.

In contrast to the plant Δ-9 desaturase, fungal and animal Δ-9 desaturases efficiently convert a wide range of saturated fatty acids with differing hydrocarbon chain lengths to monounsaturated fatty acids. The Saccharomyces cerevisiae enyzme, for example, efficiently desaturates even and odd chain fatty acyl CoA substrates from 13 carbons to 19 carbons in length. A broad functional homology exists among various Cyt b₅-dependent desaturases, as evidenced, for example, by the successful expression of the rat Δ-9 desaturase in yeast (Stukey et al., J. Biol. Chem. 2: 20144-20149, 1990).

The rat and yeast Δ-9 desaturase genes have been expressed in plants: both the rat and the yeast genes have been expressed in tobacco (Grayburn et al., BioTechnology 10: 675-678, 1992 (rat); Polashock et al., Plant Physiol. 100: 894-901, 1992 (yeast), and the yeast gene has also been expressed in tomato (Wang et al., J. Agric. Food Chem. 44: 3399-3402, 1996). The yeast Δ-9 desaturase has been shown to function in tobacco and tomato, leading to increases in the level of monounsaturated fatty acids (both 16:1 and 18:1) and other compounds derived from monounsaturated fatty acids (e.g., polyunsaturated fatty acids, hexanal, 1-hexanol, heptanal, trans-2-octenal) (Polashock et al., supra; Wang et al; supra) . Expression of the rat desaturase also led to an increase in monounsaturated 16- and 18-carbon fatty acids (Grayburn et al., supra).

From the foregoing, it can be seen that transgenic plants expressing animal or fungal Δ-9 desaturase genes can be improved in their unsaturated fatty acid composition by virtue of the activity of the foreign enzyme. Of further advantage, it has recently been discovered that some fungal Δ-9 desaturases (e.g., Saccharomyces cerevisiae) are fusion proteins comprising an intrinsic Cyt b₅ domain (Mitchell & Martin, J. Biol. Chem. 270: 29766-29772, 1995). When this gene is expressed, sufficient Cyt b₅ is produced to drive the desaturase reaction at an optimum level and is not dependent on existing plant Cyt b₅. The known animal Δ-9 desaturases do not contain this fused Cyt b₅ motif and must rely on independently-produced Cyt b₅ to provide the electrons for the reactions.

Though fungal or animal Δ-9 desaturases (e.g. the S. cerevisiae desaturase or the animal desaturases) may be expressed and functional in certain plants, their expression is likely less than optimal in plants, and expression may not even be possible in other plant species, due to several factors, including differences in codon usage and codon preference in plants as compared to fungi, and among different plant species and the presence of cryptic intron splicing signals, among others. All of these factors can lead to poor expression, or no expression, of a non-plant foreign gene in a plant cell.

Accordingly, in order to make use of non-plant fatty acid desaturases, particularly those such as the S. cerevisiae Δ-9 desaturase comprising an internal Cyt b₅ motif, a need exists to design modified desaturase-encoding DNA molecules that are customized for expression in plant cells and specific plant tissues. It would be of even greater advantage to optimize such modified DNA molecules for expression in particular plant species, such as those that are grown and harvested primarily for oils.

SUMMARY OF THE INVENTION

According to one aspect of the invention, a synthetic fatty acid desaturase gene for expression in a multi-cellular plant is provided, the gene comprising a desaturase domain and a Cyt b₅ domain, wherein the gene is customized for expression in a plant cytoplasm. In one embodiment, the synthetic gene is customized for expression in a monocotyledonous plant. In another embodiment, the synthetic gene is customized for expression in a dicotyledonous plant. In a preferred embodiment, the synthetic gene is customized for expression in a plant genus selected from the group consisting of Arabidopsis, Brassica, Phaeseolus, Oryza, Olea, Elaeis (Oil Palm) and Zea.

In a preferred embodiment of the invention, the desaturase is a cytosolic Δ-9 desaturase. The Saccharomyces cerevisiae Δ-9 desaturase is particularly preferred.

In another embodiment of the invention, the synthetic gene is customized from a naturally occurring gene comprising both a desaturase domain and a cyt b₅ domain. Alternatively, the synthetic gene is a chimeric gene comprising a desaturase domain and a heterologous cyt b₅ domain.

In another embodiment, the synthetic gene is customized from a naturally occurring gene such that the synthetic gene and the naturally occurring gene encode an identical amino acid sequence. Alternatively, the synthetic gene is customized from a naturally occurring gene such that the synthetic gene and the naturally occurring gene encode a similar and functionally conserved amino acid sequence.

In another embodiment, a naturally occurring or a synthetic gene is customized so that specific amino acid modification are made to enhance the function of the encoded protein. Examples of such modifications include changing amino acids that are subjected to phosphorylation or other post-translational modifications that may alter or regulate the activity of the Δ-9 desaturase enzyme.

In another embodiment of the invention, elements of a naturally occurring or a synthetic desaturase gene that are not essential for enzymatic function are replaced or linked with elements derived from plant ER lipid biosynthetic genes that are normally expressed in maturing seeds or other plant tissues. The improved expression of the modified gene produced by the inclusion or substitution of plant DNA sequences in the synthetic gene will result from native plant signal or control elements in those sequences that affect desaturase gene expression at one or more levels.

According to another aspect of the invention, a method is provided for constructing and customizing a bifunctional desaturase/cyt b₅ encoding gene for expression in the cytosol of a multicellular plant. The method comprises (a) providing a DNA molecule comprising a desaturase-encoding moiety operably linked to a cyt b₅-encoding moiety, said DNA molecule producing the bifunctional polypeptide in a non-customized form; (b) back-translating the polypeptide sequence using preferred codons for expression in a multicellular plant, thereby producing a back-translated nucleotide sequence; (c) analyzing the back-translated nucleotide sequence for features that could diminish or prevent expression in the plant cytoplasm, including, optionally (1) probable intron splice sites (characterized by T-rich regions); (2) plant polyadenylation signals (e.g., AATAAA); (3) polymerase II termination sequence (e.g., CAN₍₇₋₉₎AGTNNAA, where N is any nucleotide); (4) hairpin consensus sequences (e.g., UCUUCGG); and (5) the sequence-destabilizing motif ATTTA; (d) modifying the analyzed sequence to correct or remove the features that could diminish or prevent expression in the plant cytoplasm; and, optionally, (e) introducing desirable cloning features, such as restriction sites, into the sequence in a manner that does not materially affect the desired codon usage or final polypeptide sequence.

The method set forth above may be adapted by incorporating into the customized gene one or more genomic segments from plant desaturase or other ER lipid biosynthetic genes, which are determined to further optimize gene expression in plants. This method comprises (1) identifying cDNA sequences that have potential to comprise such beneficial elements, (2) creating yeast vectors expressing desaturase genes modified to contain these elements, (3) testing the vectors in a yeast expression system, (4) isolating regions from genomic DNA that are homologous to the beneficial cDNA elements, and (6) using them to construct chimeric or hybrid synthetic genes that produce functional and highly efficient desaturase activities in plant tissues.

Other features and advantages of the present invention will be better understood by reference to the drawings, detailed description and examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. GCG Pileup comparison of stearoyl-CoA desaturase protein sequences. Sequences containing a Cyt b₅ domain are indicated with a +; sequences lacking a Cyt b₅ domain are indicated with a −; sequences still in question are indicated with a ?.

FIG. 2. GCG Pileup comparison of Cytochrome b₅ protein sequences.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Various terms relating to the biological molecules of the present invention are used herein above and also throughout the specifications and claims.

The term “promoter region” refers to the 5′ regulatory regions of a gene.

The term “reporter gene” refers to genetic sequences which may be operably linked to a promoter region forming a transgene, such that expression of the reporter gene coding region is regulated by the promoter and expression of the transgene is readily assayed.

The term “selectable marker gene” refers to a gene product that when expressed confers a selectable phenotype, such as antibiotic resistance, on a transformed cell or plant.

The term “operably linked” means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements) in an expression vector.

The term “DNA construct” refers to genetic sequence used to transform plants and generate progeny transgenic plants. These constructs may be administered to plants in a viral or plasmid vector. Other methods of delivery such as Agrobacterium T-DNA mediated transformation and transformation using the biolistic process are also contemplated to be within the scope of the present invention. The transforming DNA may be prepared according to standard protocols such as those set forth in “Current Protocols in Molecular Biology”, eds. Frederick M. Ausubel et al., John Wiley & Sons, 1999.

This invention provides synthetic DNA molecules (sometimes referred to herein as “synthetic genes”) that encode a fatty acid desaturase useful for modifying the fatty acid composition of a plant. The DNA molecules describe in accordance with this invention are superior to DNA molecules currently available for this purpose, in two important respects: (1) they encode a dual-domain polypeptide (sometimes referred to herein as a “bifunctional polypeptide or protein”), one domain being the fatty acid desaturase, and the other domain being cytochrome b₅, a protein required to support the electron transfer events that enable the desaturase to function; and (2) they are customized for expression in the cytosol of plant cells, and further customized for expression in particular selected plant species.

Design of synthetic genes of the present is invention is accomplished in two broad steps. First, the two components (the desaturase-encoding component and the Cyt b₅-encoding component) are selected and linked together, if they do not occur together naturally. Second, the DNA molecule is optimized for expression in the cytosol of a plant cell, or further for expression in a particular plant species, or group of species.

With regard to the first step, it should be noted that several fungal, animal and plant species, including yeast, are now known to contain naturally-occurring genes encoding dual-domain cytoplasmic fatty acid desaturases. As mentioned above, the yeast and rat Δ-9 desaturase genes have been expressed and shown to function in plants. However, prior to the present invention, it was not appreciated that the bifunctional yeast desaturase offers a significant advantage over the single-function animal desaturase in plant cells, where the requisite Cyt b₅ is available only in small amounts, and the yeast protein can provide its own supply of Cyt b₅.

With regard to the second step—optimization for expression in the plant cytosol—it was discovered in accordance with the present invention that a non-plant desaturase-encoding gene, such as the yeast OLE 1, though expressed in some plants, may not be optimally expressed in those plants. Furthermore, the inventors have found that the yeast gene is poorly expressed in other plant species, thus highlighting the advantages obtainable by optimizing such a gene for expression in a plant cell.

Sections II-IV below describe in detail how to design and use the synthetic genes of the present invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is is not intended to limit the invention. Unless otherwise specified, general biochemical and molecular biological procedures, such as those set forth in Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory (1989) (hereinafter “Sambrook et al.”) or Ausubel et al. (eds) Current Protocols in Molecular Biology, John Wiley & Sons (1999) (hereinafter “Ausubel et al.”) are used.

II. Design and Construction of the Synthetic DNA Molecules

A. Selection of component DNA segments

This invention contemplates the use of the following source DNAs, which are thereafter modified for expression in plants, if necessary:

1. naturally occurring genes or cDNAs that encode dual domain polypeptides comprising a desaturase domain and a Cyt b₅ domain;

2. chimeric genes in which a desaturase-encoding sequence from one source (e.g., the desaturase domain of a dual domain fungal Δ-9 desaturase, or the single domain rat desaturase), is linked to a Cyt b₅-encoding sequence from a different source (e.g., a plant);

3. chimeric genes in which a sequence that encodes a fragment of a naturally occurring plant Cyt b₅ (e.g. the heme binding fold, or residues that comprise the electron donor or acceptor sites, or residues that act as membrane targeting or retention signals, or residues that act to stabilize the protein in the plant cytoplasmic environment) is substituted for homologous regions in the cytochrome b₅ domain of a dual domain polypeptide such as the yeast Δ-9 desaturase; and

4. chimeric genes in which elements that encode the essential enzymatic domains from one source (e.g. a native or synthetic gene derived from a fungal Δ-9 desaturase) are linked to elements derived from native plant desaturases that enhance transcription, mRNA processing, mRNA stability, protein folding and maturation, membrane targeting or retention, or protein stability.

Naturally occurring genes or cDNAs that encode dual domain desaturase/Cyt b₅ proteins have been identified in several fungal species, including Saccharomyces cerevisiae, Pichia augusta, Histoplasma capsulatum and Cryptococcus curvatus (See FIG. 1). Naturally occurring genes or cDNA=s that encode independent, diffusible Cyt b₅ proteins have been identified in several plant species, including Nicotiana tabacum (tobacco), Oryza sativa (rice), Cuscuta reflexa (southern Asian dodder), Arabidopsis thaliana, Brassica oleracea and Olea europaea (olive). A N-terminal Cyt b₅ domain of a Δ-6 desaturase has also been identified in the plant Borago officinalis, and in the Saccharomyces cerevisiae FAH1 gene that encodes a very long chain fatty acid hydroxylase. Genes or cDNAs from these species, as well as DNA from any other species identified in the future as encoding such a dual domain protein, are contemplated for use in the synthetic genes of the present invention.

In a preferred embodiment, the yeast OLE1 gene is used. This embodiment is described in detail in Example 1.

The second strategy involves linking a DNA segment encoding a fatty acid desaturase from one source with a Cyt b₅ domain from another source. In a preferred embodiment, this chimeric gene is fashioned after the naturally-occurring dual function genes discussed above. That is, the Cyt b₅ domain and the desaturase domain are situated in the same positions respective to each other as is found in the naturally occurring genes (see, e.g., Mitchell & Martin, J. Biol. Chem. 270: 29766-29772, 1996).

The chimeric dual-domain proteins of the invention are prepared by recombinant DNA methods, in which DNA sequences encoding each domain are operably linked together such that upon expression, a fusion protein having the desaturase and Cyt b₅ functions described above is produced. As defined above, the term “operably linked” means that the DNA segments encoding the fusion protein are assembled with respect to each other, and with respect to an expression vector in which they are inserted, in such a manner that a functional fusion protein is effectively expressed. The selection of appropriate promoters and other 5′ and 3′ regulatory regions, as well as the assembly of DNA segments to form an open reading frame, employs standard methodology well. known to those skilled in the art.

Thus, preparing the chimeric DNAs of the invention involves selecting DNA sequences encoding each of the aforementioned components and operably linking the respective sequences together in an appropriate vector. The sequences are thereafter expressed to produce the dual-function protein.

Genes or cDNAs that encode single-function cytoplasmic Δ-9 fatty acid desaturases have been identified in a diverse array of procaryotic and eucaryotic species, including insects, fungi and mammals, but not plants (FIG. 1). Genes or cDNAs from any of these species, as well as DNA from any other species identified in the future as encoding a fatty acid desaturase, are contemplated for use in the synthetic genes of the present invention.

In preferred embodiments, desaturase-encoding genes from eucaryotes, most preferably fungi or mammals, are used. In a particularly preferred embodiment, a DNA encoding the rat stearoyl CoA desaturase is used. This DNA has been successfully expressed in tobacco, and accordingly is expected to be useful as part of a chimeric desaturase/Cyt b₅ gene of the present invention.

Genes or cDNAs that encode Cyt b₅ proteins have also been identified in a diverse array of eucaryotic species, including insects, fungi, mammals and plants. Genes or cDNAs from any of these species, as well as DNA from any other species identified in the future as encoding a Cyt b₅ protein, are contemplated for use in the synthetic genes of the present invention.

In preferred embodiments, Cyt b₅-encoding genes or cDNAs from plants are used. These DNAs are preferred because they naturally comprise the codon usage preferred in plants, so require little, if any, of the modification steps described below for non-plant genes. Particularly preferred, if available, are Cyt b₅-encoding DNAs from the same plant species (or group of species) to be transformed with the chimeric gene. For instance, synthetic chimeric genes constructed for transformation of Brassica species might comprise a stearoyl CoA-encoding domain from rat and a Cyt b₅ domain from Brassica (see FIGS. 1 and 2 for specific sources). This chimeric DNA would require optimization for expression in Brassica only in the desaturase domain.

With respect to the naturally-occurring dual domain-encoding genes, as well as the chimeric genes discussed above, it will be appreciated that the DNA molecules can be prepared in a variety of ways, including DNA synthesis, cloning, mutagenesis, amplification, enzymatic digestion, and similar methods, all available in the standard literature. Additionally, certain DNA molecules can be obtained by access to public repositories, such as the American Type Culture Collection. Alternatively, DNA molecules that are not readily available, and/or for which sequence information is not available, can be isolated from biological sources using standard hybridization methods and homologous probes that are available.

B. Optimization for expression in plants

The second step in designing the synthetic DNA molecules of the invention is to customize (i.e. optimize) their sequence for expression in the plant cytoplasm. This is accomplished by performing one or more of the steps listed below on the coding sequence of the above described non-plant (or chimeric) desaturase/Cyt b₅-encoding DNA molecules.

1. From the peptide sequence encoded by the DNA, back translate using an appropriate plant codon usage table, making certain in particular that the most preferred translation termination codon is used.

2. Visually, or with the aid of computer software, analyze the back-translated nucleotide sequence for features that could diminish or prevent expression in the plant cytoplasm. Such features include: (1) probable intron splice sites (characterized by T-rich regions); (2) plant polyadenylation signals (e.g., AATAAA); (3) polymerase II termination sequence (e.g., CAN₍₇₋₉₎AGTNNAA, where N is any nucleotide); (4) hairpin consensus sequences (e.g., UCUUCGG); and (5) the sequence-destabilizing motif ATTTA (Shah & Kamen, Cell 4: 659-667, 1986). These features have been described in the art (U.S. Pat. No. 5,500,365 to Fischhoff et al.; U.S. Pat. No. 5,380,831 to Adang et al.).

3. Modify the back-translated sequence in light of any “problem” sequences identified in step 2. Note that this step may require the introduction of codons that are not the most preferred, but instead are second or third-most preferred, in order to eliminate the more problematic sequences identified in step 2.

4. Introduce desirable cloning features, such as restriction sites, into the sequence in a manner that does not materially affect the desired codon usage or final polypeptide sequence.

The aforementioned optimization procedure can be performed so that the final polypeptide sequence is identical to the initial polypeptide sequence, even though the underlying nucleotide sequence has been modified. This is a preferred embodiment of the invention. However, it is entirely feasible to modify the initial sequence such that the final sequence is not identical to the initial sequence, either by virtue of amino acid substitutions, insertions or deletions. The more that is known about the structure/function relationship in a particular desaturase protein, the more liberties can be taken in modifying the protein sequence during the DNAoptimization process. For instance, the present inventors have shown that the entire “coiled coil” domain of the yeast OLE1 gene can be deleted, and the protein remains functional. Thus, it appears that OLE1 can tolerate significant modification in the encoded protein without losing its biological activity.

Codon usage tables for a variety of plants, including general plant codon usage tables, tables for dicots, tables for monocots, and tables for particular species, are widely available. Some of these are reproduced in Example 1 below. One good location to access such tables is the website:

http://biochem.octago.ac.nz.800/Transterm/codons.html.

In an exemplary embodiment of the present invention, the above process is applied to the coding sequence of the yeast OLE1 gene, which encodes a cytoplasmically expressed dual-domain protein comprising a Δ-9 fatty acid desaturase domain and a Cyt b₅ domain. Optimization of the OLE1 gene for expression in Arabidopsis and related species is described in detail in Example 1.

In another preferred embodiment, the coding sequence of the rat stearoyl CoA desaturase is modified for expression in plants according to the methods described above. The modified sequence is operably linked to a coding sequence for a Cyt b₅ domain, preferably from a plant, and most preferably from Brassica. In this regard, it has been shown that expression of this rat desaturase in tobacco produces a functional protein that increases the 16:1 fatty acid content of plant tissues. Splice site prediction analysis of the rat desaturase reveals that there are no plant intron-like sequences within the open reading frame. However, codon usage analysis reveals that this desaturase possesses a number of codons that are not optimal for expression in plants, particularly Arabidopsis or Brassica.

In another preferred embodiment, the protein coding sequences of the modified vectors described above are further modified to increase desaturase activity. This is done by altering specific amino acids in the encoded protein that control desaturase activity through post-translational modifications. These modifications are presumed to increase the level of desaturase activity in the host plant by stabilizing the desaturase protein or by increasing catalytic activity of the desaturase. Post translational modifications such as protein phosphorylation or dephosphorylation have been shown to alter activity of a number of enzymes by a number of different mechanisms. These include increasing or decreasing enzyme activity or protein stability, or changing the intracellular location of the enzyme. An examination of a wide range of Δ-9 desaturase enzymes reveals the existence of a number of highly conserved potential phosphorylations sites that could serve as sequences that regulate desaturase activity. These are shown in bold face on the pile-up diagram in FIG. 3 and are summarized in Table 1 of Example 3. The high degree of homology between these sites suggests that these sequences may also be recognized by host plant phosphorylating or dephosphorylating enzymes. If phosphorylation of an amino acid within one of the sites increases the activity of the desaturase, the nucleic acid sequence corresponding to that amino acid can be altered to encode a negatively charged amino acid at that site to permanently increase the activity of the protein in the host. If phosphorylation of an amino acid within the site reduces the activity of the desaturase enzyme, the nucleic acid sequence can be altered to replace that amino acid with a neutral amino acid that will permanently increase the activity of the enzyme.

In another preferred embodiment, elements of the genes in the modified vectors described above are further modified and improved by the linkage or substitution of sequences derived from native plant ER lipid biosynthetic genes. Those sequences contain elements that improve the desaturase activity by increasing the efficiency of gene expression, intracellular protein targeting and/or enzyme stability. This is done by identifying elements of the engineered desaturase gene that can be replaced or linked with elements of a plant gene without significantly affecting the desired activity or specificity of the resulting enzyme. Genes and cDNAs that encode ER lipid biosynthetic enzymes from Brassica, Arabidopsis, Nicotiana tabacum, Borage, maize, sunflower and soybeans, as well as similar plant genes from any other species that are identified in the future, are contemplated for use in the synthetic genes of the present invention.

In connection with the aforementioned embodiment, but not limited thereto, it is particularly useful in many cases to pre-test constructs of the invention in a yeast expression system, in order to eliminate constructs that work poorly before taking the more labor- and time-intensive step of testing them in plants. Accordingly, this step may be incorporated into the methods described herein.

III. Construction of Vectors for Transforming Plant Nuclei, and Production of Transgenic Plants Expressing Synthetic Genes of the Invention

The synthetic genes of the present invention are intended for use in producing transgenic plants that optimally express a dual-function desaturase/Cyt b₅ protein in the cytoplasm of plant cells. Transformation of plant nuclei to produce transgenic plants may be accomplished according to standard methods known in the art. These include, but are not limited to, Agrobacterium vectors, PEG treatment of protoplasts, biolistic DNA delivery, UV laser microbeam, gemini virus vectors, calcium phosphate treatment of protoplasts, electroporation of isolated protoplasts, agitation of cell suspensions with microbeads coated with the transforming DNA, direct DNA uptake, liposome-mediated DNA uptake, and the like. Such methods have been published in the art. See, e.g., Methods for Plant Molecular Biology, Weissbach & Weissbach eds., Academic Press, Inc. (1988); Methods in Plant Molecular Biology, Schuler & Zielinski, eds., Academic Press, Inc. (1989); Plant Molecular Biology Manual, Gelvin Schilperoort, Verma, eds., Kluwer Academic Publishers, Dordrecht (1993); and Methods in Plant Molecular Biology—A Laboratory Manual, Maliga, Klessig, Cashmore, Gruissem & Varner, eds., Cold Spring Harbor Press (1994).

The method of transformation depends upon the plant to be transformed. The biolistic DNA delivery method is useful for nuclear transformation, and is a preferred method for practice of this invention. In another embodiment of the invention, Agrobacterium vectors are used to advantage for efficient transformation of plant nuclei.

In a preferred embodiment, the synthetic gene is introduced into plant nuclei in Agrobacterium binary vectors. Such vectors include, but are not limited to, BIN19 (Bevan, Nucl. Acids Res., 12: 8711-8721, 1984) and derivatives thereof, the pBI vector series (Jefferson et al., EMBO J., 6: 3901-3907, 1987), and binary vectors pGA482 and pGA492 (An, Plant Physiol., 81: 86-91, 1986). A new series of Agrobacterium binary vectors, the pPZP family, is preferred for practice of the present invention. The use of this vector family for plant transformation is described by Svab et al. in Methods in Plant Molecular Biology—A Laboratory Manual, Maliga, Klessig, Cashmore, Gruissem and Varner, eds., Cold Spring Harbor Press (1994).

Using an Agrobacterium binary vector system for transformation, the synthetic gene of the invention is linked to a nuclear drug resistance marker, such as kanamycin or gentamycin resistance. Agrobacterium-mediated transformation of plant nuclei is accomplished according to the following procedure:

(1) the gene is inserted into the selected Agrobacterium binary vector;

(2) transformation is accomplished by co-cultivation of plant tissue (e.g., leaf discs) with a suspension of recombinant Agrobacterium, followed by incubation (e.g., two days) on growth medium in the absence of the drug used as the selective medium (see, e.g., Horsch et al., Science 227: 1229-1231, 1985);

(3) plant tissue is then transferred onto the selective medium to identify transformed tissue; and

(4) identified transformants are regenerated to intact plants.

It should be recognized that the amount of expression, as well as the tissue specificity of expression of the synthetic genes in transformed plants can vary depending on the position of their insertion into the nuclear genome. Such position effects are well known in the art; see Weising et al., Ann. Rev. Genet., 22: 421-477 (1988). For this reason, several nuclear transformants should be regenerated and tested for expression of the synthetic gene.

IV. Uses of the Synthetic Genes and Transgenic Plants Expressing Those Genes

The synthetic desaturase genes of the invention and transgenic plants expressing those genes can be used for several agriculturally beneficial purposes. For instance, they can be used in oil-producing crops (e.g., corn, soybean, sunflower, rapeseed) to increase the overall percentages of monounsaturated fatty acids in those oils, thereby improving their health-promoting qualities. In this regard, the production of transgenic rapeseed plants (Brassica napus) is of particular interest in this invention. Example 1 describes a synthetic yeast desaturase gene modified for expression in Arabidopsis. Because the codon usage of Brassica is very similar to that of Arabidopsis, it is expected that the synthetic gene described in Example 1 will be as well expressed in Brassica as it is in Arabidcopsis.

Another use for the synthetic genes of the invention is to modify the flavors of certain fruit or vegetable crops. It has already been shown that expression of the un-modified yeast Δ-9 desaturase gene in tomato results in alterations in fatty acid composition and fatty acid-derived flavor compounds (Wang et al., 1996, supra). The synthetic, plant-optimized version of this gene is expected to function similarly, and also to be more efficiently expressed in plant cells.

Another use for the synthetic genes of the invention is to facilitate the formation of omega-5 anacardic acids, a class of secondary compounds derived from the Δ-9 desaturation of 14:0 in pest-resistant geraniums (Schultz et al., Proc. Natl. Acad. Sci. USA, 93: 877-885, 1996). It has been shown that formation of these compounds proceeds from the expression of Δ9 desaturase activity resulting in the formation of Δ9 14:1. Subsequent elongation of these molecules leads to the formation of omega-5 22:1 and 24:1 in the trichome exudate that leads to pest resistance against spider mites and aphids.

Another use for the synthetic genes of the invention are in the modification of membrane lipid fatty acyl composition to alter the properties of the cytoplasmic and plasma membranes of the cell. These may affect functions such membrane associated activities that are associated with membrane functions such as signal transduction, endocytosis or exocytotic events, entry of fungal or viral pathogens into the cell, and temperature or environmentally caused stress that causes physical changes in the fluid properties of the plasma membrane or internal cell membranes. Plants defective in desaturases have been reported (Somerville and Browse, supra). These mutant plants contain higher than normal levels of saturated fatty acids that may lower membrane fluidity under normal growing conditions. Thus the effects of temperature on these plants involved high temperature tolerance as opposed to chilling tolerance. These studies yielded interesting information that has relevance to temperature stress in general. A mutant of Arabidopsis deficient in 16:0 desaturation (Hugly et al, Plant Physiol. 90: 1134-1142) for example, has been shown to appear and grow normally at non-stressful temperatures. Under high temperature conditions, however, the mutant performs better than controls in growth and biosynthetic studies. Higher temperature stability was also noted in pea thylakoids following catalytic hydrogenation (Thoman et al. Biochem. Biophys. Acta 849: 131-140, 1986).

The following examples are provided to describe the invention in greater detail. They are intended to illustrate, not to limit, the invention.

EXAMPLE 1 Modification of the Saccharomyces cerevisiae OLE1 Gene for Expression in Arabidopsis and Related Species

When introduced into tobacco and tomato plants, the yeast Δ-9 desaturase gene (OLE1) was shown to desaturate palmitate and stearate, thereby reducing the levels of saturated fatty acids in triglycerides (Polashock et al., supra; Wang et al., supra) . However, it was unclear whether optimum expression of the OLE1 gene occurred in those species, and expression in other plant species has been less than optimum. For example, the present inventors have found that the level of expression of the OLE1 gene in tobacco (Polashock, et. al., Plant Physiol. 100:894-901, 1992) and Arabidopsis varies in different plant tissues and is generally poor in tobacco, and Arabidopsis seeds. Similarly, data from other investigators indicate that expression of OLE1 in rapeseed (Brassica napus) seeds is also poor (U.S. Pat. No. 5,777,201, to Poutre, et al.).

Differential expression of heterologous genes in plants can be caused by several factors. It is often due to the presence of cryptic intron splicing signals. Thus, it is possible that the multiple banding patterns observed in northern blots of OLE1-transformed tobacco (Polashock et al., supra) are due to splicing of the OLE1 mRNA.

In plants, the mRNA splicing mechanism is less well defined than in mammalian or yeast systems. There is some conservation of the 5′ and 3′ splicing signals but there is no conserved internal splice signal. However, with the accumulation of plant genomic DNA sequence data, it is now becoming possible to predict with some accuracy where intron splicing will occur (Hebsgaard, S. M., P. G. Korning, N. Tolstrup, J. Engelbrecht, P. Rouze and S. Brunak, Nucleic Acids Research 24(17): 3439-3452, 1996). In fact, computer programs that predict splice sites have now been developed (the “PlantNetGene” server for splice site predictions: http://www.cbs.dtu.dk/NetPlantGene.html). From these sources, it appears that plant introns are typically identified as T rich sequences.

Another factor affecting expression of foreign genes in plants is codon preference. It is now well known that preference for certain codons exist among different phyla, classes, families, genera and species. Accordingly, by modifying a DNA sequence so that it uses codons preferred in a particular organism, expression of that sequence can be optimized.

Other factors affecting the expression of foreign genes in plants include the presence of putative polyadenylation signals, hairpin cleavage consensus motifs, polymerase II termination sequences and the Shaw-Kamen sequence pattern ATTTA.

This example describes the design and construction of “pl-ole1”, a modified Saccharomyces cerevisiae OLE1 gene optimized for expression in Arabidopsis and other plant species.

The nucleotide sequence of the Saccharomyces cerevisiae OLE1 gene coding sequence has been described in U.S. Pat. No. 5,057,419 to Martin et al. (incorporated by reference herein) and is set forth below for convenience as SEQ ID NO:1 (open reading frame starts at +11). The S. cerevisiae Δ-9 desaturase amino acid sequence encoded by OLE1 is set forth as SEQ ID NO:2.

I. Design of pl-ole1

To modify OLE1 for optimum expression in plants, the OLE1 sequence was first analyzed for cryptic plant splice signals, using the PlantNetGene server for splice site predictions. This analysis identified a number of “high confidence” intron splice signals in the OLE1 sequence. These are shown below (positions correspond to position numbers in SEQ ID NO:1).

5′-3′ 5′-3′ Position Strand Confidence exon {circumflex over ( )}intron Donor splice site, direct strand: (Start ATG = +1)  397 + 1.00 GCTCTCTCTG {circumflex over ( )}GTAAAGTACC 1052 + 0.85 CTATTAAGTG {circumflex over ( )}GTACCAATAC 1074 + 1.00 CCCAACTAAG {circumflex over ( )}GTTATCATCT Accentor splice site, direct strand: 500 + 0.86 GGTCTCACAG {circumflex over ( )}ATCTTACTCC

Next, the CLE1 peptide sequence (SEQ ID NO:2) was back-translated using an Arabidopsis thaliana codon usage table, as shown below. Codon usage in Arabidopsis and several other plant species, including Brassica napus, Phaseolus vulgaris and Zea mays is very similar, as can be seen by a comparison with the respective codon usage tables of those species, also shown below (the codon usage table of Saccharomyces cerevisiae is shown for comparison; codon usage tables taken from Ahttp://biochem.otago.ac.nz:800/Transterm/codons.html).

AmAcid Codon Number /1000 Fraction Arabidopsis thaliana. Gly GGG 6027.00 10.31 0.14 Gly GGA 15393.00 26.32 0.37 Gly GGT 14890.00 25.46 0.35 Gly GGC 5654.00 9.67 0.13 Glu GAG 19825.00 33.90 0.51 Glu GAA 18672.00 31.93 0.49 Asp GAT 20862.00 35.67 0.65 Asp GAC 11061.00 18.91 0.35 Val GTG 10414.00 17.81 0.26 Val GTA 5145.00 8.80 0.13 Val GTT 16157.00 27.63 0.41 Val GTC 8156.00 13.95 0.20 Ala GCG 5361.00 9.17 0.13 Ala GCA 10552.00 18.04 0.25 Ala GCT 18782.00 32.12 0.45 Ala GCC 7249.00 12.40 0.17 Arg AGG 6684.00 11.43 0.22 Arg AGA 10280.00 17.58 0.34 Ser AGT 7369.00 12.60 0.16 Ser AGC 6399.00 10.94 0.14 Lys AAG 20436.00 34.94 0.55 Lys AAA 16882.00 28.87 0.45 Asn AAT 11658.00 19.93 0.47 Asn AAC 12987.00 22.21 0.53 Met ATG 14817.00 25.34 1.00 Ile ATA 6571.00 11.24 0.21 Ile ATT 13028.00 22.28 0.41 Ile ATC 11855.00 20.27 0.38 Thr ACG 4346.00 7.43 0.14 Thr ACA 8703.00 14.88 0.28 Thr ACT 10909.00 18.65 0.36 Thr ACC 6720.00 11.49 0.22 Trp TGG 6868.00 11.74 1.00 End TGA 652.00 1.11 0.44 Cys TGT 5641.00 9.65 0.58 Cys TGC 4154.00 7.10 0.42 End TAG 252.00 0.43 0.17 End TAA 591.00 1.01 0.40 Tyr TAT 8052.00 13.77 0.47 Tyr TAC 8965.00 15.33 0.53 Leu TTG 11727.00 20.05 0.22 Leu TTA 6361.00 10.88 0.12 Phe TTT 11703.00 20.01 0.47 Phe TTC 13066.00 22.34 0.53 Ser TCG 4830.00 8.26 0.10 Ser TCA 9033.00 15.45 0.19 Ser TCT 13022.00 22.27 0.28 Ser TCC 6214.00 10.63 0.13 Arg CGG 2531.00 4.33 0.08 Arg CGA 3142.00 5.37 0.10 Arg CGT 5680.00 9.71 0.19 Arg CGC 2100.00 3.59 0.07 Gln CAG 9564.00 16.35 0.47 Gln CAA 10908.00 18.65 0.53 His CAT 7466.00 12.77 0.58 His CAC 5415.00 9.26 0.42 Leu CTG 5669.00 9.69 0.11 Leu CTA 5350.00 9.15 0.10 Leu CTT 14395.00 24.61 0.27 Leu CTC 9751.00 16.67 0.18 Pro CCG 4676.00 8.00 0.17 Pro CCA 9131.00 15.61 0.33 Pro CCT 10732.00 18.35 0.39 Pro CCC 3331.00 5.70 0.12 Brassica napus Gly GGG 730.00 11.21 0.13 Gly GGA 2042.00 31.37 0.36 Gly GGT 1952.00 29.99 0.35 Gly GGC 892.00 13.70 0.16 Glu GAG 2119.00 32.55 0.55 Glu GAA 1764.00 27.10 0.45 Asp GAT 1895.00 29.11 0.56 Asp GAC 1478.00 22.70 0.44 Val GTG 1231.00 18.91 0.28 Val GTA 493.00 7.57 0.11 Val GTT 1624.00 24.95 0.36 Val GTC 1124.00 17.27 0.25 Ala GCG 615.00 9.45 0.13 Ala GCA 1167.00 17.93 0.24 Ala GCT 2028.00 31.15 0.42 Ala GCC 1056.00 16.22 0.22 Arg AGG 697.00 10.71 0.22 Arg AGA 996.00 15.30 0.32 Ser AGT 736.00 11.31 0.15 Ser AGC 803.00 12.34 0.17 Lys AAG 2243.00 34.46 0.55 Lys AAA 1817.00 27.91 0.45 Asn AAT 1058.00 16.25 0.37 Asn AAC 1811.00 27.82 0.63 Met ATG 1538.00 23.63 1.00 Ile ATA 669.00 10.28 0.20 Ile ATT 1271.00 19.52 0.37 Ile ATC 1461.00 22.44 0.43 Thr ACG 563.00 8.65 0.15 Thr ACA 1059.00 16.27 0.28 Thr ACT 1154.00 17.73 0.30 Thr ACC 1073.00 16.48 0.28 Trp TGG 798.00 12.26 1.00 End TGA 69.00 1.06 0.37 Cys TGT 517.00 7.94 0.50 Cys TGC 509.00 7.82 0.50 End TAG 33.00 0.51 0.18 End TAA 83.00 1.28 0.45 Tyr TAT 792.00 12.17 0.38 Tyr TAC 1283.00 19.71 0.62 Leu TTG 1051.00 16.14 0.20 Leu TTA 508.00 7.80 0.09 Phe TTT 1003.00 15.41 0.39 Phe TTC 1562.00 23.99 0.61 Ser TCG 475.00 7.30 0.10 Ser TCA 856.00 13.15 0.18 Ser TCT 1147.00 17.62 0.24 Ser TCC 799.00 12.27 0.17 Arg CGG 219.00 3.36 0.07 Arg CGA 297.00 4.56 0.09 Arg CGT 659.00 10.12 0.21 Arg CGC 275.00 4.22 0.09 Gln CAG 1188.00 18.25 0.50 Gln CAA 1168.00 17.94 0.50 His CAT 651.00 10.00 0.49 His CAC 672.00 10.32 0.51 Leu CTG 592.00 9.09 0.11 Leu CTA 579.00 8.89 0.11 Leu CTT 1416.00 21.75 0.26 Leu CTC 1208.00 18.56 0.23 Pro CCG 542.00 8.33 0.15 Pro CCA 1180.00 18.13 0.33 Pro CCT 1281.00 19.68 0.36 Pro CCC 527.00 8.10 0.15 Phaseolus vulgaris Gly GGG 371.00 13.30 0.15 Gly GGA 771.00 27.64 0.32 Gly GGT 817.00 29.29 0.34 Gly GGC 441.00 15.81 0.18 Glu GAG 912.00 32.69 0.54 Glu GAA 767.00 27.50 0.46 Asp GAT 776.00 27.82 0.55 Asp GAC 625.00 22.41 0.45 Val GTG 661.00 23.70 0.36 Val GTA 174.00 6.24 0.09 Val GTT 653.00 23.41 0.36 Val GTC 346.00 12.40 0.19 Ala GCG 180.00 6.45 0.09 Ala GCA 528.00 18.93 0.26 Ala GCT 791.00 28.36 0.39 Ala GCC 553.00 19.82 0.27 Arg AGG 324.00 11.61 0.29 Arg AGA 325.00 11.65 0.29 Ser AGT 317.00 11.36 0.14 Ser AGC 353.00 12.65 0.15 Lys AAG 1054.00 37.78 0.60 Lys AAA 697.00 24.99 0.40 Asn AAT 555.00 19.90 0.42 Asn AAC 782.00 28.03 0.58 Met ATG 567.00 20.33 1.00 Ile ATA 274.00 9.82 0.20 Ile ATT 539.00 19.32 0.40 Ile ATC 548.00 19.65 0.40 Thr ACG 166.00 5.95 0.11 Thr ACA 362.00 12.96 0.24 Thr ACT 480.00 17.21 0.32 Thr ACC 490.00 17.57 0.33 Trp TGG 342.00 12.26 1.00 End TGA 34.00 1.22 0.44 Cys TGT 145.00 5.20 0.39 Cys TGC 229.00 8.21 0.61 End TAG 22.00 0.79 0.28 End TAA 22.00 0.79 0.28 Tyr TAT 400.00 14.34 0.40 Tyr TAC 597.00 21.40 0.60 Leu TTG 543.00 19.47 0.24 Leu TTA 184.00 6.60 0.08 Phe TTT 458.00 16.42 0.43 Phe TTC 601.00 21.55 0.57 Ser TCG 149.00 5.34 0.06 Ser TCA 416.00 14.91 0.18 Ser TCT 606.00 21.72 0.26 Ser TCC 501.00 17.96 0.21 Arg CGG 71.00 2.55 0.06 Arg CGA 76.00 2.72 0.07 Arg CGT 169.00 6.06 0.15 Arg CGC 158.00 5.66 0.14 Gln CAG 437.00 15.67 0.48 Gln CAA 470.00 16.85 0.52 His CAT 298.00 10.68 0.46 His CAC 355.00 12.73 0.54 Leu CTG 351.00 12.58 0.15 Leu CTA 184.00 6.60 0.08 Leu CTT 569.00 20.40 0.25 Leu CTC 452.00 16.20 0.20 Pro CCG 147.00 5.27 0.08 Pro CCA 694.00 24.88 0.37 Pro CCT 664.00 23.80 0.36 Pro CCC 352.00 12.62 0.19 Zea mays Gly GGG 2466.00 15.07 0.19 Gly GGA 2186.00 13.36 0.17 Gly GGT 2607.00 15.93 0.20 Gly GGC 5499.00 33.61 0.43 Glu GAG 7364.00 45.01 0.72 Glu GAA 2823.00 17.25 0.28 Asp GAT 3425.00 20.93 0.37 Asp GAC 5740.00 35.08 0.63 Val GTG 4365.00 26.68 0.38 Val GTA 916.00 5.60 0.08 Val GTT 2516.00 15.38 0.22 Val GTC 3644.00 22.27 0.32 Ala GCG 3698.00 22.60 0.24 Ala GCA 2517.00 15.38 0.16 Ala GCT 3602.00 22.01 0.24 Ala GCC 5481.00 33.50 0.36 Arg AGG 2500.00 15.28 0.27 Arg AGA 1199.00 7.33 0.13 Ser AGT 1170.00 7.15 0.10 Ser AGC 2776.00 16.97 0.24 Lys AAG 7241.00 44.25 0.79 Lys AAA 1969.00 12.03 0.21 Asn AAT 1946.00 11.89 0.33 Asn AAC 3939.00 24.07 0.67 Met ATG 4071.00 24.88 1.00 Ile ATA 1014.00 6.20 0.13 Ile ATT 2099.00 12.83 0.28 Ile ATC 4403.00 26.91 0.59 Thr ACG 1890.00 11.55 0.22 Thr ACA 1620.00 9.90 0.19 Thr ACT 1757.00 10.74 0.21 Thr ACC 3236.00 19.78 0.38 Trp TGG 1994.00 12.19 1.00 End TGA 199.00 1.22 0.45 Cys TGT 770.00 4.71 0.28 Cys TGC 1963.00 12.00 0.72 End TAG 121.00 0.74 0.28 End TAA 120.00 0.73 0.27 Tyr TAT 1303.00 7.96 0.27 Tyr TAC 3440.00 21.02 0.73 Leu TTG 1807.00 11.04 0.13 Leu TTA 582.00 3.56 0.04 Phe TTT 1697.00 10.37 0.29 Phe TTC 4082.00 24.95 0.71 Ser TCG 1620.00 9.90 0.14 Ser TCA 1592.00 9.73 0.14 Ser TCT 1792.00 10.95 0.15 Ser TCC 2746.00 16.78 0.23 Arg CGG 1505.00 9.20 0.16 Arg CGA 610.00 3.73 0.06 Arg CGT 1018.00 6.22 0.11 Arg CGC 2562.00 15.66 0.27 Gln CAG 4280.00 26.16 0.72 Gln CAA 1626.00 9.94 0.28 His CAT 1378.00 8.42 0.36 His CAC 2431.00 14.86 0.64 Leu CTG 4069.00 24.87 0.29 Leu CTA 904.00 5.52 0.07 Leu CTT 2415.00 14.76 0.17 Leu CTC 4079.00 24.93 0.29 Pro CCG 2642.00 16.15 0.29 Pro CCA 2152.00 13.15 0.23 Pro CCT 2102.00 12.85 0.23 Pro CCC 2344.00 14.33 0.25 Saccharomyces cerevisiae Gly GGG 18129.00 6.18 0.12 Gly GGA 32850.00 11.20 0.22 Gly GGT 66575.00 22.69 0.45 Gly GGC 28821.00 9.82 0.20 Glu GAG 57100.00 19.46 0.30 Glu GAA 133513.00 45.51 0.70 Asp GAT 111120.00 37.88 0.65 Asp GAC 58642.00 19.99 0.35 Val GTG 32144.00 10.96 0.20 Val GTA 35470.00 12.09 0.22 Val GTT 63678.00 21.71 0.39 Val GTC 33136.00 11.30 0.20 Ala GCG 18402.00 6.27 0.11 Ala GCA 47728.00 16.27 0.30 Ala GCT 58916.00 20.08 0.37 Ala GCC 35917.00 12.24 0.22 Arg AGG 27990.00 9.54 0.21 Arg AGA 61524.00 20.97 0.47 Ser AGT 42499.00 14.49 0.16 Ser AGC 29298.00 9.99 0.11 Lys AAG 89539.00 30.52 0.42 Lys AAA 124327.00 42.38 0.58 Asn AAT 106379.00 36.26 0.60 Asn AAC 71659.00 24.43 0.40 Met ATG 61216.00 20.87 1.00 Ile ATA 53773.00 18.33 0.28 Ile ATT 88869.00 30.29 0.46 Ile ATC 49422.00 16.85 0.26 Thr ACG 24131.00 8.23 0.14 Thr ACA 52363.00 17.85 0.31 Thr ACT 58260.00 19.86 0.34 Thr ACC 35998.00 12.27 0.21 Trp TGG 30707.00 10.47 1.00 End TGA 1901.00 0.65 0.30 Cys TGT 23942.00 8.16 0.62 Cys TGC 14448.00 4.93 0.38 End TAG 1421.00 0.48 0.23 End TAA 2985.00 1.02 0.47 Tyr TAT 55441.00 18.90 0.57 Tyr TAC 42016.00 14.32 0.43 Leu TTG 79248.00 27.01 0.28 Leu TTA 77691.00 26.48 0.28 Phe TTT 78451.00 26.74 0.59 Phe TTC 53809.00 18.34 0.41 Ser TCG 25856.00 8.81 0.10 Ser TCA 55962.00 19.08 0.21 Ser TCT 69019.00 23.53 0.26 Ser TCC 41460.00 14.13 0.16 Arg CGG 5414.00 1.85 0.04 Arg CGA 9166.00 3.12 0.07 Arg CGT 18429.00 6.28 0.14 Arg CGC 7924.00 2.70 0.06 Gln CAG 36018.00 12.28 0.31 Gln CAA 78385.00 26.72 0.69 His CAT 40211.00 13.71 0.64 His CAC 22609.00 7.71 0.36 Leu CTG 31503.00 10.74 0.11 Leu CTA 39789.00 13.56 0.14 Leu CTT 36697.00 12.51 0.13 Leu CTC 16401.00 5.59 0.06 Pro CCG 15796.00 5.38 0.12 Pro CCA 51725.00 17.63 0.41 Pro CCT 39402.00 13.43 0.31 Pro CCC 20387.00 6.95 0.16

For each amino acid, the new pl-ole1 gene was designed the codon most preferred in Arabidopsis, with the following exceptions:

1. The codon for glutamine CAG was switched to CAA. Though the codon preference for glutamine is the same for both CAG and CAA in Arabidopsis, CAA was used since the AG motif is part of the 3′ intron splice signal.

2. In OLE1, there are regions of high leucine/valine amino acid usage (e.g., between positions 322 to 571 of the nucleotide sequence are codons coding for 11 leucines and 7 valines). These regions correspond to the OLE1 protein transmembrane domains. If the most preferred codons in Arabidopsis (CTT and GTT, respectively) were used, the region would take on the characteristics of a plant intron, i.e., high T content, thereby introducing a number of highly probable 5′ splice sites, which could not be removed without altering the amino acid sequence. Accordingly, a mixture of alternative codons was used for these amino acids. Similar changes were also applied to two other regions of OLE1 (positions 781 to 900 and positions 1081 to 1140).

Next, a search for problematic sequences, such as putative polyadenylation signals, hairpin cleavage consensus motifs, ATTTA motifs or concatamers thereof, was conducted. Such sequences are described in detail in U.S. Pat. No. 5,380,831 to Adang et al. (incorporated by reference herein). This search identified one hairpin cleavage consensus motif, CTTCGG, at position 553-559 of SEQ ID NO:1, which was removed by changing TTC to TTT (both encoding phenylalanine).

Next, a BamHI site and translation initiation consensus were added to the 5′ end of the OLE1 coding sequence (M. Kozak, J. Biol. Chem. 266(30): 19867-19870, 1991). An XbaI and a BamHI site were added to the 3′ end of the coding sequence. A PacI site was introduced into the same position as the original S. cerevisiae OLE1 PacI site (within the cytochrome b₅ domain), in order to provide a convenient restriction site for construction of this and other synthetic OLE1 genes. Other convenient restriction sites, which enable modular construction of synthetic OLE1 genes, are inherent within the final sequence of the new pl-ole1 gene.

Finally, the termination codon was checked against a stop codon consensus database, “TransTerm” (Dalphin et al., Nucl. Acids Res. 25(1): 246-247, 1997). The existing termination sequence, TGAT, appeared suitable for use in Arabidopsis, and so was not altered.

II. Construction of pl-ole1

The rebuilt pl-ole1 nucleotide sequence was constructed commercially (Operon Technologies, Inc.). The plasmid containing the rebuilt gene was designated pAMCM013. The pl-ole1 nucleotide sequence is set forth below as SEQ ID NO:3 (open reading frame starts at +11). This sequence encodes SEQ ID NO:2, but differs from the S. cerevisiae OLE1 gene (SEQ ID NO:1) in the following respects (summarized from above):

1. Arabidopsis thaliana codon usage; CAG switched to CAA for glutamine;

2. Translation initiation consensus added;

3. Hairpin removed;

4. Several (but not all) PlantNetGene predicted splice sites removed;

5. Eleven leucines changed from CTT to CTC, and 7 valines changed from GTT to GTG in positions 322-571, which corresponds to a plant intron-like region; similar changes made in regions 781-900 and 1081-1140; valine at position 432 retained as GTT to maintain Psp1406I site;

6. Certain leucine and valine codons were altered so that the same codons would not appear adjacent to others;

7. Intron acceptor site at position 1047 altered;

8. Restriction sites added to allow modular construction; PsP1406I site removed at position 1441; and

9. PacI site introduced at position 1362; an introduced NgoMI site at position 867 removed.

A gap alignment of SEQ ID NO: 1 (top) and SEQ ID NO: 3 (bottom) is shown below:

Gap alignment of wild type and rebuilt OLE1 sequences. Percent Similarity: 79.871 Percent Identity: 79.871

         .         .         .         .         .    1 TACAACAAAGATGCCAACTTCTGGAACTACTATTGAATTGATTGACGACC 50      |||  ||||| ||||||||||||||||| ||  | || || || |    1 ggatccaacaATGCCTACTTCTGGAACTACTATCGAGCTTATCGATGATC 50          .         .         .         .         .   51 AATTTCCAAAGGATGACTCTGCCAGCAGTGGCATTGTCGACGAAGTCGAC 100 |||| || |||||||| |||||      ||| || || || || || ||   51 AATTCCCTAAGGATGATTCTGCTTCTTCTGGAATCGTTGATGAGGTTGAT 100          .         .         .         .         .  101 TTAACGGAAGCTAATATTTTGGCTACTGGTTTGAATAAGAAAGCACCAAG 150  | || || ||||| ||  | ||||||||  | || ||||| || || ||  101 CTTACTGAGGCTAACATCCTTGCTACTGGACTTAACAAGAAGGCTCCTAG 150          .         .         .         .         .  151 AATTGTCAACGGTTTTGGTTCTTTAATGGGCTCCAAGGAAATGGTTTCCG 200 ||| || ||||| || || ||| | ||||| || ||||| |||||||| |  151 AATCGTTAACGGATTCGGATCTCTTATGGGATCTAAGGAGATGGTTTCTG 200          .         .         .         .         .  201 TGGAATTCGACAAGAAGGGAAACGAAAAGAAGTCCAATTTGGATCGTCTG 250 | || ||||| |||||||||||||| |||||||| ||  | ||| | ||  201 TTGAGTTCGATAAGAAGGGAAACGAGAAGAAGTCTAACCTTGATAGACTT 250          .         .         .         .         .  251 CTAGAAAAGGACAACCAAGAAAAAGAAGAAGCTAAAACTAAAATTCACAT 300 || || ||||| |||||||| || || || ||||| ||||| || || ||  251 CTTGAGAAGGATAACCAAGAGAAGGAGGAGGCTAAGACTAAGATCCATAT 300          .         .         .         .         .  301 CTCCGAACAACCATGGACTTTGAATAACTGGCACCAACATTTGAACTGGT 350 ||| || ||||| |||||| | || |||||||| |||||| | ||||||  301 CTCTGAGCAACCTTGGACTcTCAACAACTGGCATCAACATCTcAACTGGC 350          .         .         .         .         .  351 TGAACATGGTTCTTGTTTGTGGTATGCCAATGATTGGTTGGTACTTCGCT 400 | |||||||| || || ||||| ||||| ||||| || ||||||||||||  351 TcAACATGGTgCTcGTcTGTGGAATGCCTATGATCGGATGGTACTTCGCT 400          .         .         .         .         .  401 CTCTCTGGTAAAGTACCTTTGCATTTAAACGTTTTCCTTTTCTCCGTTTT 450 |||||||| ||||| ||| | ||| | ||||||||||| ||||| || ||  401 CTcTCTGGAAAaGTgCCTCTcCATCTcAACGTTTTCcTcTTCTCTGTcTT 450          .         .         .         .         .  451 CTACTACGCTGTCGGTGGTGTTTCTATTACTGCCGGTTACCATAGATTAT 500 |||||||||||| || || || ||||| ||||| || ||||||||| | |  451 CTACTACGCTGTTGGAGGAGTgTCTATCACTGCTGGATACCATAGACTcT 500          .         .         .         .         .  501 GGTCTCACAGATCTTACTCCGCTCACTGGCCATTGAGATTATTCTACGCT 550 ||||||| ||||||||||| ||||| |||||  | ||| | |||||||||  501 GGTCTCATAGATCTTACTCTGCTCATTGGCCTCTTAGACTcTTCTACGCT 550          .         .         .         .         .  551 ATCTTCGGTTGTGCTTCCGTTGAAGGGTCCGCTAAATGGTGGGGCCACTC 600 ||||| || |||||||| ||||| || || ||||| |||||||| || ||  551 ATCTTtGGATGTGCTTCTGTTGAGGGATCTGCTAAGTGGTGGGGACATTC 600          .         .         .         .         .  601 TCACAGAATTCACCATCGTTACACTGATACCTTGAGAGATCCTTATGACG 650 ||| ||||| || ||| | |||||||||||  | ||||||||||| || |  601 TCATAGAATCCATCATAGATACACTGATACTCTTAGAGATCCTTACGATG 650          .         .         .         .         .  651 CTCGTAGAGGTCTATGGTACTCCCACATGGGATGGATGCTTTTGAAGCCA 700 || | ||||| || |||||||| || ||||||||||||||| | |||||  651 CTAGAAGAGGACTTTGGTACTCTCATATGGGATGGATGCTTCTTAAGCCT 700          .         .         .         .         .  701 AATCCAAAATACAAGGCTAGAGCTGATATTACCGATATGACTGATGATTG 750 || || || |||||||||||||||||||| || |||||||||||||||||  701 AACCCTAAGTACAAGGCTAGAGCTGATATCACTGATATGACTGATGATTG 750          .         .         .         .         .  751 GACCATTAGATTCCAACACAGACACTACATCTTGTTGATGTTATTAACCG 800 ||| || ||||||||||| ||||| ||||||||| | ||| |  | || |  751 GACTATCAGATTCCAACATAGACATTACATCtTgCTcATGCTcCTTACTG 800          .         .         .         .         .  801 CTTTCGTCATTCCAACTCTTATCTGTGGTTACTTTTTCAACGACTATATG 850 ||||||| || || ||||| |||||||| ||||| |||||||| || |||  801 CTTTCGTgATCCCTACTCTcATCTGTGGATACTTCTTCAACGATTACATG 850          .         .         .         .         .  851 GGTGGTTTGATCTATGCCGGTTTTATTCGTGTCTTTGTCATTCAACAAGC 900 || ||  | ||||| || || || ||  | || || ||||| ||||||||  851 GGAGGACTcATCTACGCTGGATTCATCAGAGTgTTCGTcATCCAACAAGC 900          .         .         .         .         .  901 TACCTTTTGCATTAACTCCATGGCTCATTACATCGGTACCCAACCATTCG 950 ||| || || || ||||| ||||||||||||||||| || ||||| ||||  901 TACTTTCTGTATCAACTCTATGGCTCATTACATCGGAACTCAACCTTTCG 950          .         .         .         .         .  951 ATGACAGAAGAACCCCTCGTGACAACTGGATTACTGCCATTGTTACTTTC 1000 |||| |||||||| ||| | || |||||||| ||||| || |||||||||  951 ATGATAGAAGAACTCCTAGAGATAACTGGATCACTGCTATCGTTACTTTC 1000          .         .         .         .         . 1001 GGTGAAGGTTACCATAACTTCCACCACGAATTCCCAACTGATTACAGAAA 1050 || || || |||||||||||||| || || ||||| |||||||| ||||| 1001 GGAGAGGGATACCATAACTTCCATCATGAGTTCCCTACTGATTAtAGaAA 1050          .         .         .         .         . 1051 CGCTATTAAGTGGTACCAATACGACCCAACTAAGGTTATCATCTATTTGA 1100 |||||| ||||||||||||||||| || ||||| || |||||||| |||| 1051 CGCTATCAAGTGGTACCAATACGATCCTACTAAaGTgATCATCTACtTgA 1100          .         .         .         .         . 1101 CTTCTTTAGTTGGTCTAGCATACGACTTGAAGAAATTCTCTCAAAATGCT 1150 ||||| | || || || || |||||  | ||||| ||||||||||| ||| 1101 CTTCTCTcGTgGGACTTGCTTACGATCTcAAGAAGTTCTCTCAAAACGCT 1150          .         .         .         .         . 1151 ATTGAAGAAGCCTTGATTCAACAAGAACAAAAGAAGATCAATAAAAAGAA 1200 || || || ||  | || |||||||| |||||||||||||| || ||||| 1151 ATCGAGGAGGCTCTTATCCAACAAGAGCAAAAGAAGATCAACAAGAAGAA 1200          .         .         .         .         . 1201 GGCTAAGATTAACTGGGGTCCAGTTTTGACTGATTTGCCAATGTGGGACA 1250 |||||||||||| ||||| || ||| | |||||| | || |||||||| | 1201 GGCTAAGATtAAtTGGGGACCTGTTCTTACTGATCTTCCTATGTGGGATA 1250          .         .         .         .         . 1251 AACAAACCTTCTTGGCTAAGTCTAAGGAAAACAAGGGTTTGGTTATCATT 1300 | ||||| ||| | |||||||||||||| ||||||||  | |||||||| 1251 AGCAAACTTTCCTTGCTAAGTCTAAGGAGAACAAGGGACTTGTTATCATC 1300          .         .         .         .         . 1301 TCTGGTATTGTTCACGACGTATCTGGTTATATCTCTGAACATCCAGGTGG 1350 ||||| || ||||| || || ||||| || |||||||| ||||| || || 1301 TCTGGAATCGTTCATGATGTTTCTGGATACATCTCTGAGCATCCTGGAGG 1350          .         .         .         .         . 1351 TGAAACTTTAATTAAAACTGCATTAGGTAAGGACGCTACCAAGGCTTTCA 1400  || ||||||||||| |||||  | || ||||| ||||| ||||||||| 1351 AGAGACTttaATtAAGACTGCTCTTGGAAAGGATGCTACTAAGGCTTTCT 1400          .         .         .         .         . 1401 GTGGTGGTGTCTACCGTCACTCAAATGCCGCTCAAAATGTCTTGGCTGAT 1450  ||| || || ||| | || || || || |||||||| ||  | |||||| 1401 CTGGAGGAGTTTACAGACATTCTAACGCTGCTCAAAACGTGCTTGCTGAT 1450          .         .         .         .         . 1451 ATGAGAGTGGCTGTTATCAAGGAAAGTAAGAACTCTGCTATTAGAATGGC 1500 |||||||| ||||||||||||||   ||||||||||||||| |||||||| 1451 ATGAGAGTTGCTGTTATCAAGGAGTCTAAGAACTCTGCTATCAGAATGGC 1500          .         .         .         .         . 1501 TAGTAAGAGAGGTGAAATCTACGAAACTGGTAAGTTCTTTTAAGCATCAC 1550 |  ||||||||| || |||||||| ||||| |||||||| | | |   | 1501 TTCTAAGAGAGGAGAGATCTACGAGACTGGAAAGTTCTTCTGAtctagag 1550 1551 ATTAC 1555   | | 1551 gatcc 1555

The pl-ole1 synthetic gene contains no intron-like regions, or predicted splice sites within its sequence. Moreover, comparing the codon usage of Arabidopsis with that of Brassica napus, Phaseolus vulgaris or Zea mays, with the exception of cystein (a rare amino acid that comprises 1.7% of all Arabidopsis codons, and occurs 4 times (0.8%) in OLE1), the sequence contains no rare codons for any of those species. The codon usage of pl-ole1 is particularly similar to the preferred usage of Brassica napus. Accordingly, pl-ole1 is expected to be particularly well expressed in all those species, and well expressed in any plant species.

An alternative version of pl-ole1, referred to herein as pl-ole1-2, was also constructed. This synthetic gene was modified only in specific codons identified as high frequency splicing signals. It was discovered that this construct is expressed equally as well as pl-ole1 in Arabidopsis.

EXAMPLE 2 Vacuum Infiltration Transformation of Arabidopsis thaliana with pl-ole1

A modification of a transformation protocol of Pam Green (http://www.bch.msu.edu/pamgreen/vac.html) was used for the transformation of A. thaliana with pl-ole1. The protocol was adapted from protocols by Nicole Bechtold and Andrew Bent. This protocol gives very good results, with 95% of all infiltrated plants giving rise to transformants, and a transformant in up to 1 in 25 seeds.

Protocol

1. Seeds of Arabidopsis thaliana ecotype Columbia were sown in lightweight plastic pots prepared in the following way: mound Arabidopsis soil mixture into 3 to 4 inch pots, saturate soil with Arabidopsis fertilizer, add more soil so that it is rounded about 0.5 above the edge.

2. Plants were grown under conditions of 16 hours light/8 hours dark at 20° C., fertilizing with Arabidopsis fertilizer once a week from below, adding about 0.5 L to each flat. After 4-6 weeks, plants were considered ready for vacuum infiltration when primary inflorescence was 10-15 cm tall and the secondary inflorescences appeared at the rosette. The bolts were clipped back and 2 to 3 days was allowed for them to regrow before infiltration.

3. in the meantime, the construct was transformed into Agrobacterium tumefaciens strain (LBA4404). When plants were ready to transform, a 50 mL culture of LB medium containing 50 mg/L kanamycin and 50 mg/L of streptomycin was inoculated with a 1 mL overnight starter culture.

4. Cultures were grown overnight at 28° C. with shaking. The culture was pelleted, the supernatant removed, and the pellet resuspended in 250 ml of infiltration medium to OD600>0.8. Infiltration medium (1 liter) comprised 2.2 g MS salts, 1×B5 vitamins, 50 g sucrose, 0.5 g MES, pH to 5.7 with KOH, 0.044=B5M benzylaminopurine, 200=B5L Silwet L-77 (OSl Specialties).

5. The resuspended culture was placed in a magenta jar inside a large bell jar. Pots containing plants to be infiltrated were inverted into the solution so that the entire plant was covered, including rosette, but none of the soil was submerged.

6. A vacuum of 400 mm Hg (about 17 inches) was drawn. Once the vacuum level was reached, the suction was closed and the plants allowed to remain under vacuum for five minutes. The vacuum was then quickly released. The pots were briefly drained, then placed on their sides in a tray, which was covered with a humidome to maintain humidity. The next day, the plants were removed to the growth room, the pots uncovered and set upright. Plants infiltrated with different constructs were kept separated in different trays thereafter.

7. Plants were allowed to grow under the same conditions as before. Plants were staked individually as the bolts grew. When plants were finished flowering, water was gradually reduced, then eliminated to allow the plants to dry out. Seeds were harvested from each plant individually.

8. Large selection plates were prepared: 4.3 g/L MS salts; 1×E5 vitamins (optional); 1% sucrose; 0.5 g/L MES pH to 5.7 with KOH; 0.8% phytagar—Autoclaved, then added antibiotics (35 μg/mL kanamycin and 250 μg/mL of carbenicillin) and 150×15 mm plates were poured.

9. Plates were dried well in the sterile hood before plating—20-30 minutes with the lids open was usually sufficient.

10. For each plant, up to 100 μL of seeds (approximately 2500 seeds) was sterilized and plated out individually. Seeds were sterilized as follows: 1 min in 70% ethanol, 7 minutes in 50% bleach/0.02% Triton X-100 with vortexing, 6 rinses in sterile distilled water. Seeds were resuspended in 2 mL sterile 0.1% agarose and poured onto large selection plates as if plating phage. Plates were tilted so seeds were evenly distributed, and allowed to sit 10-15 minutes, during which time the liquid soaked into the medium. Plates were sealed with Parafilm and placed in a growth room.

11. After 7 to 10 days, transformants were visible as dark green plants. These were transferred onto “hard selection” plates (100×15 mm plates with same recipe as selection plates but with 1.5% phytagar) to eliminate any pseudo-resistants, then replaced in the growth room.

12. After 10 to 14 days, the plants possessed at least two sets of true leaves. At this point, plants were transferred to soil, covered with plastic, and moved to a growth chamber with normal conditions. They were typically kept covered for several days.

References

Bechtold N, Ellis J, Pelletier G (1998) Methods Mol Biol. 82: 259-266.

Bent A, Kunkel B N, Dahlbeck D, Brown K L, Schmidt R, Giraudat J, Leung J, Staskawicz B J (1994) Science 265: 1856-1860.

Koncz C, Schell J (1986) Mol. Gen. Genet. 204: 383-396.

Solutions

1000×B5 Vitamins (10 mL)

1000 mg myo-inositol

100 mg thiamine-HCl

10 mg nicotinic acid

10 mg pyridoxine-HCl

Dissolve in ddH2O and store at −20° C.

Arabidopsis Fertilizer (10 liters)

50 mL 1M KNO3

25 mL 1M KPO4 (pH 5.5)

20 mL 1M MgSO4

20 mL 1M Ca(NO3)2

5 mL 0.1M Fe.EDTA

10 mL micronutrients (see below)

Dissolve in ddH2O and store at room temperature

Arabidopsis Micronutrients (500 mL)

70 mL 0.5M boric acid

14 mL 0.5M MnCl2

2.5 mL 1M CuSO4

1 mL 0.5M ZnSO4

1 mL 0.1M NaMoO4

1 mL 5M NaCl

0.05 mL 0.1M CuCl2

Dissolve in ddH2O and store at room temperature

EXAMPLE 3 Customizing OLE1 to Express Post-Translational Modifications

After determining the optimized codon preferences of OLE1 mRNA (or mRNA derived from another fungal or animal desaturase) for high level expression in the host plant, specific amino acids that are involved in the post-translational control of enzyme activity or stability are altered to maximize the catalytic activity of the expressed enzyme. There are a number of protein kinase and/or phosphorylase consensus sequences that are highly conserved in the fungal and animal desaturases. These are shown below. First is shown a table of aligned potential phosphorylation sites in desaturases. Next is shown a pileup of Δ-9 fatty acid desaturases. PROSITE analysis of these desaturases predicts a number of potential phosphorylation sites, highlighted by bold underlined characters.

TABLE 2 Aligned Potential phosphorylation sites in desaturases. Phosphorylation sites are indicated with respect to amino acid positions in the Olelp protein coding sequence. Abbreviations, S = serine, T = Threonine, A = Alanine, PKC — protein kinase C like; CK-2 — casein kinase II like; CAMP — cAMP activated kinase (PKA) like; np., not predicted as a phosphorylation site. Residue Olelp Olelp Phosphory- Fungal/ position Residue sequence lation type b5 Animal Insect 166 S SHR PKC mixed, all S, N.P., A S or A SHR 169 T np PKC S or A all TYK SYK or SYK 191 S SAK PKC all S all D all A 206 T np CK-2 all T all S all S 208 T TLRD CK-2 all T all T all T 215 A np PKC A or V all S all A 323 T TPRD CK-2 T or S S or np np 351 R np CK-2 all R all S all K 383 S KKFS CAMP S or np all S S but np

Pileup of Δ-9 fatty acid desaturases showing potential phosphorylation sites

1                                                   50 Rat ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜MPAHM LQE.ISSSY. Mouse ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜MPAHM LQE.ISSSY. Sheep ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜MPAHL LQEEISSSY. Pig ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜SSY. Human ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜MPAHL LQDDISSSY. Hamster ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜MPGHL LQEEMTSSYT Drosophila ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜MPP NAQAGAQSIS Moth ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ C. elegans ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜MTVKTRSN IAKKIEKDGG S. cerevisiae MPTSGTTIEL IDDQFPKDDS ASSGIVDEVD LTEANILATG LNKKAPRIVN P. angusta ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ H. capsulatum ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ M. rouxii ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ C. curvatus ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ C. merolae ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ MTAKVESKVR EEEKGSNPST 51                                                 100 Rat TTTTTITEPP SGNLQNGREK MKKVPLYLEE DI.....RPE MREDIHDPSY Mouse TTTTTITAPP SG...NEREK VKTVPLHLEE DI.....RPE MKEDIHDPTY Sheep TTTTTITAPP SRVLQNGGGK LEKTPLYLEE DI.....RPE MRDDIYDPNY Pig TTTTTITAPS SRVLQNGGGK SEKTPQYVEE DI.....RPE MKDDIYDPTY Human TTTTTITAPP PGVLQNGGDK LETMPLYLED DI.....RPD IKDDIYDPTY Hamster TTTTTITEPP SESLQ..... .KTVPLYLEE DI.....RPE MKEDIYDPSY Drosophila DSLIAAASAA ADAGQSPTKL QEDSTGVLFE CD.....VET TDGGLVKDTT Moth ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜MPPQG QTGGSWVLYE TD.....AVN TDTD..APVI C. elegans PETQYLAVDP NEIIQLQEES KKVVPKCLPA RLPTAACKAS QENGECQKTV S. cerevisiae GFGSLMGSKE MVSVEFDKKG NEKKSNLDRL LEKDNQEKEE AKTKIH.ISE P. angusta ˜˜˜˜˜MGTKS MTDVTAEEL. ..SKDSVAMM LAKDRELKNK YLKQKH.ISE H. capsulatum ˜˜˜˜˜˜˜˜MA LNEAPTASPV AETAAGGKDV VTDAARRPNS EPKKVH.ITD M. rouxii ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜MSN IATLTSTART KTESMKPPLP KTKMPP.LFD C. curvatus ˜MSASTKQAS TTVAQPSGKP VTNVIDPERD DFIVPDNYVT RTVENM.KML C. merolae AAADDSGAVI PTLKPRPKPA VEPLEREGVE FDPQRGLVFE KTRSSKWMSE 101                                                150 Rat QDEEGPPPKL EYVWRNIILM ALLHVGALYG ITL.IPSSKV YTLLWGIFYY Mouse QDEEGPPPKL EYVWRNIILM VLLHLGGLYG IIL.VPSCKL YTALFGIFYY Sheep QDKEGPKPKL EYVWRNIILM GLLHLGALYG ITL.IPTCKI YTFLWVLFYY Pig QDKEGPQGKL EYVWRNIILM SLLHLGALYG IIL.IPTCKI YTLLWAFAYY Human KDKEGPSPKV EYVWRNIILM SLLHLGALYG ITL.IPTCKF YTWLWGVFYY Hamster QDEEGPPPKL EYVWRNIILM ALLHLGALYG LVL.VPSSKV YTLLWAFVYY Drosophila VMKKAEKRLL KLVWRNIIAF GYLHLAALYG AYLMVTSAKW QTCILAYFLY Moth VPPSAEKREW KIVWRNVILM GMLHIGGVYG AYLFLTKAMW LTDLFAFFLY C. elegans FLEIVIPYKM EIVWRNVALF AALHFAAAIG LYQLIFEAKW QTVIFTFLLY S. cerevisiae QPWTLNNWHQ HLNWLNMVLV CGMPMIGWYF ALSGKVPLHL NVFLFSVFYY P. angusta QPWTWENWHR HINWLNFILV LAVPFAG..L ISTKWVPLKL HTFVTAVILY H. capsulatum TPITLANWHK HISWLNVTLT IAIPIYG..L VQAYWVPLHL KTALWAVVYY M. rouxii QPVTSKNWTK FVNWPQAILL CVTPLIALYG IFT..TELTK KTLIWSWIYY C. curvatus PPVTWRNLHK NIQWISFLAL TIPPAMAIYG LCT..VPVQT KTFIWSVVYY C. merolae KELNELPLLQ RINWLS.TSI IFTPLIGT.L IGIWFVPLQR KTLVLAIVTY 151                                                200 Rat LISALGITAG AHRLWSHRTY KARLPLRIFL IIANTMAFQN DVYEWARDHR Mouse MTSALGITAG AHRLWSHRTY KARLPLRIFL IIANTMAFQN DVYEWARDHR Sheep VISALGITAG VHRLWSHRTY KARLPLRVFL IIANTMAFQN DVFEWSRDHR Pig LLSAVGVTAG AHRLWSHRTY KARLPLRVFL IIANTMAFQN DVYEWARDHR Human FVSALGITAG AHRLWSHRSY KARLPLRLFL IIANTMAFQN DVYEWARDHR Hamster VISIEGIGAG VHRLWSHRTY KARLPLRIFL IIANTMAFQN DVYEWARDHR Drosophila VISGLGITAG AHRLWAHRSY KAKWPLRVIL VIFNTIAFQD AAYHWARDHR Moth LCSGLGITAG AHRLWAHKSY KARLPLRLLL TLFNTLAFQD AVIDWARDHR C. elegans VFGGFGITAG AHRLWSHKSY KATTPMRIFL MILNNIALQN DVIEWARDHR S. cerevisiae AVGGVSITAG YHRLWSHRSY SAHWPLRLFY AIFGCASVEG SAKWWGHSHR P. angusta CFGGISITAG YHRHWAHRAY DCKLPVKIFF ALFGASAVEG SIKMWGHQHR H. capsulatum FMTGLGITAG YHRLWAHCSY SATLPLKIYL AAVGGGAVEG SIRWWARGHR M. rouxii FITGLGITAG YHRMWSHRAY RGTDLLRWFM SFAGAGAVEG SIYWWSRGHR C. curvatus FITGLGITAG YHRLWAHRSY NASKPLQYFL ALCGAGSVQG SIRWWSRGHR C. merolae FCCGLGITGG YHRLWSHRSY EAHWLVQVIL ACFGAAAFEG SARYWCRLHR 201                                                250 Rat AHHKFSETHA DPHNSRRGFF FSHVGWLLVR KHPAVKEKGG KLDMSDLKAE Mouse AHHKFSETHA DPHNSRRGFF FSHVGWLLVR KHPAVKEKGG KLDMSDLKAE Sheep AHHKFSETDA DPHNSRRGFF FSHVGWLLVR KHPAVREKGA TLDLSDLRAE Pig AHHKFSETDA DPHNSRRGFF FSHVGWLLVR KHPAVKEKGG LLNMSDLKAE Human AHHKFSETHA DPHNSRRGFF FSHVGWLLVR KHPAVKEKGS TLDLSDLEAE Hamster AHHKFSETYA DPHNSRRGFF FSHVGWLLVR KHPAVKEKGG KLDMSDLKAE Drosophila VHHKYSETDA DPHNATRGFF FSHVGWLLCK KHPEVKAKGK GVDLSDLRAD Moth MHHKYSETDA DPHNATRGFF FSHVGWLLVR KHPQIKAKGH TIDLSDLKSD C. elegans CHHKWTDTDA DPHNTTRGFF FAHMGWLLVR KHPQVKEQGA KLDMSDLLSD S. cerevisiae IHHRYTDTLR DPYDARRGLW YSHMGWMLLK PNP...KYKA RADITDMTDD P. angusta VHHRYTDTPR DPYDAKRGFW YSHMGWMLLV PNP...RYKA RADISDLLDD H. capsulatum AHHRYTDTDK DPYSVRKGLL YSHIGWMVMK QNP...KRIG RTEITDLNED M. rouxii AHHRWTDTDK DPYSAHRGFF FSHFGWMLVQ RPK...NRIG YADVADLKAD C. curvatus AHHRYTDTKL DPYSAHEGFW HAHMGWMLI. KPR...GKIG VADISDLSKN C. merolae AHHRYVDSDR DPYAVEKGFW YAHLWWMVFK LPR...QRQG RVDITDLNAN 251                                                300 Rat KLVMFQRRYY KPGLLLMCFI LPTLVPWYCW GETFLHSLFV STFLRYTLVL Mouse KLVMFQRRYY KPGLLLMCFI LPTLVPWYCW GETFVNSLFV STFLRYTLVL Sheep KLVMFQRRYY KPGVLLLCFI LPTLVPWYLW GESFQNSLFF ATFLRYAVVL Pig KLVMFQRRYY KPGILLMCFI LPTIVPWYCW GEAFPQSLFV ATFLRYAIVL Human KLVMFQRRYY KPGLLMMCFI LPTLVPWYFW GETFQNSVFV ATFLRYAVVL Hamster KLVMFQRRYY KPAILLMCFI LPTFVPWYFW GEAFVNSLCV STFLRYALVL Drosophila PILMFQKKYY MILMPIACFI IPTVVPMYAW GESFMNAWFV ATMFRWCFIL Moth PILRFQKKYY LTLMPLICFI LPSYIPT.LW GESAFNAFFV CSIFRYVYVL C. elegans PVLVFQRKHY FPLVILCCFI LPTIIPVYFW KETAFIAFYT AGTFRYCFTL S. cerevisiae WTIRFQHRHY ILLMLLTAFV IPTLICGYFF ND.YMGGLIY AGFIRVFVIQ P. angusta WVVRVQHRHY LLLMVVMAFL FPAVLTHYLF ND.FWGGFIY AGLLRAVVIQ H. capsulatum PVVVWQHRNY LKVVIFMGIV FPMLVSGLGW GD.WFGGFIY AGILRIFFVQ M. rouxii HVVAFQHKYY PYFALGMGFI FPTLVAGLGW GD.FRGGYFY AGVLRLCFVH C. curvatus PVVKWQHNNY VALLFFMGLA FPTLVAGLGW GD.WWGGLFF AGAARLVFVH C. merolae PILRFQHRYY LQIAILFSFV IPLTISTLGW GD.FWGGLVY ACLGRMLFVQ 301                                                350 Rat NATWLVNSAA HLYGYRPYDK NIQSRENILV SLGSVGEGFH NYHHAFPYDY Mouse NATWLVNSAA HLYGYRPYDK NIQSRENILV SLGAVGEGFH NYHHTFPFDY Sheep NATWLVNSAA HMYGYRPYDK TINPRENILV SLGAVGEGFH NYHHTFPYDY Pig NATWLVNSAA HLYGYRPYDK TISPRENILV SLGAVGEGFH NYHHTFPYDY Human NATWLVNSAA HLFGYRPYDK NISPRENILV SLGAVGEGFH NYHHSFPYDY Hamster NATWLVNSAA HLYGYRPYDK NIDPRENALV SLGCLGEGFH NYHHAFPYDY Drosophila NVTWLVNSAA HKFGGRPYDK FINPSENISV AILAFGEGWH NYHHVFPWDY Moth NVTWLVNSAA HLWGSKPYDK NINPVETRPV SLVVLGEGFH NYHHTFPWDY C. elegans HATWCINSAA HYFGWKPYDS SITPVENVFT TIAAVGEGGH NFHHTFPQDY S. cerevisiae QATFCINSLA HYIGTQPFDD RRTPRDNWIT AIVTFGEGYH NFHHEFPTDY P. angusta QATFCVNSLA HWIGEQPFDD RRTPRDHVLT ALVTFGEGYH NFHHEFPSDY H. capsulatum QATFCVNSLA HWLGDQPFDD RNSPRDHIVT ALVTLGEGYH NFHHEFPSDY M. rouxii HATFCVNSLA HYLGESTFDD HNTPRDSWVT ALVTMGEGYH NFHHQFPQDY C. curvatus HSTFCVNSLA HWLGETPFDN KHTPKDHFIT ALVTVGEGYH NFHHQFPMDF C. merolae QSTFCVNSLA HWWGEQTFSR RHTSYDSVIT ALVTLGEGYH NFHHEFPHDY 351                                                400 Rat SASEY.RWHI NFTTFFIDCM AALGLAYDRK KVSKAAVLAR IKRTGDGSHK Mouse SASEY.RWHI NFTTFFIDCM AALGLAYDRK KVSKATVLAR IKRTGDGSHK Sheep SASEY.RWHI NFTTFFIDCM AAIGLAYDRK KVSKAAVLGR MKRTGEESYK Pig SASEY.RWHI NLTTFFIDCM AALGLAYDRK KVSKAAIL˜˜ ˜ ˜˜˜˜˜˜˜˜˜ Human SASEY.RWHI NFNTFFIDWM AALGLTYDRK KVSKAAILAR IKRTGDGNYK Hamster SASEY.RWHI NFTTFFIDCM AALGLAYDRK KVSKAAVLAR IKRTGDGSCK Drosophila KTAEFGKYSL NFTTAFIDFF AKIGWAYDLK TVSTDIIKKR VKRTGDGTHA Moth KTAELGDYSL NFTKMFIDFM ASIGWAYDLK TVSTDVIQKR VKRTGDGSHA C. elegans RTSEYS.LKY NWTRVLIDTA AALGLVYDRK TACDEIIGRQ VSNHGCDIQR S. cerevisiae RNA.IKWYQY DPTKVIIYLT SLVGLAYDLK KFSQNAIEEA LIQQEQKKIN P. angusta RNA.LKWYQY DPTKVVIYLL SKVGLAYNLK KFSQNAIDQG ILQQQQKKLD H. capsulatum RNA.IEWHQY DPTKWTIWIW KQLGLAYDLK QFRANEIEKG RVQQLQKKID M. rouxii RNA.IKFGQY DPTKWKIIVL SWFGLAYELK QFPTNEVTKG RLFMEEKRIQ C. curvatus RNA.IKWYQY DPTKWFIWTM AQLGLASHLK KFPDNEIKKG QYTMKLMQLQ C. merolae RNG.VVWYHW DPTKWVIRLL SWAGLAWHLV RFPRNELVKA RLQVRQEILD 401                                                450 Rat SS*˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ Mouse SS˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ Sheep SG˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ Pig ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ Human SG˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ Hamster SG˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ Drosophila TWGWGDVDQP KEEIE.DAVI THKKSE˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ Moth VWGWDDHEVH QEDKKLAAII NPEKTE˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ C. elegans GKSIM˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ S. cerevisiae KKKAKINWGP VLTDLPMWDK QTFLAKS.KE NKGLVIISGI VHDVSGYISE P. angusta RMRAKLNWGP QLSELPVWDK STFFEKA.KE QKGLVIISGI VHDCANFLTE H. capsulatum QRRAKLDWGI PLEQLPVIEW DDYVDQA.KN GRGLIAIAGV VHDVTDFIKD M. rouxii AQKAKLSYGT PLKDLPIYTW EEYQSLVLND NKKWVLIEGV LYDVEEFMKE C. curvatus EQSEKLEWPK HSNDLPVISW EDFQA..ESK TRALIAVHGF IHDCSSFIED C. merolae EAKKRVDWGK PIESLPVTTW KDVQRLAKEE NRLLVVIEGI VHDCTRFKVQ 451                                                500 Rat ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ Mouse ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ Sheep ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ Pig ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ Human ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ Hamster ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ Drosophila ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ Moth ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ C. elegans ˜˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ ˜ ˜˜˜˜˜˜˜˜˜ S. cerevisiae HPGGETLIKT ALGKDATKAF SGGVYRHSNA AQNVLADMRV AVIKESKNSA P. angusta HPGGQALLKT SFGKDATMAF NGGVYAHSNA AHNLLATMRV AVIRDGGANG H. capsulatum HPGGKAMINS GIGKDATAMF NGGVYNHSNA AHNQLSTMRV GVIRGGCEVE M. rouxii HPGGMKYLST AVGKDMTTAF NGGIYNHSNG TRNLLTSLRV GVLRNGMQV. C. curvatus HPGGAHLIKR AIGTDSTTAF FGGVYDHSNA AHNLLAMMRV GVLDGGMEVE C. merolae HPGGQRILEF WNVRDATQAF NGDVYNHTKA ARNLLAHLRV AQLKEIYEPE

Protein kinase (specifically cAMP- and cGMP-dependent) phosphorylation sites.

There have been a number of studies relative to the specificity of cAMP- and cGMP-dependent protein kinases (Fremisco J. R. et al., J. Biol. Chem. 255:4240-4245, 1980; Glass D. B., Smith S. B., J. Biol. Chem. 258:14797-14803, 1983; Glass D. B. et al., J. Biol. Chem. 261:2987-2993, 1986). Both types of kinases appear to share a preference for the phosphorylation of serine or threonine residues found close to at least two consecutive N-terminal basic residues. It is important to note that there are quite a number of exceptions to this rule. However, the consensus pattern is as follows: [RK] (2)-x-[ST], where S or T is the phosphorylation site.

Protein kinase C phosphorylation site.

In vivo, protein kinase C exhibits a preference for the phosphorylation of serine or threonine residues found close to a C-terminal basic residue (Woodget J. R. et al., Eur. J. Biochem. 161:177-184, 1986;. Kishimoto A. et al., J. Biol. Chem. 260:12492-12499, 1985). The presence of additional basic residues at the N- or C-terminus of the target amino acid enhances the Vmax and Km of the phosphorylation reaction. The consensus pattern is: [ST]-x-[RK] where S or T is the phosphorylation site.

Casein kinase II phosphorylation site.

Casein kinase II (CK-2) is a protein serine/threonine kinase whose activity is independent of cyclic nucleotides and calcium. CK-2 phosphorylates many different proteins. The substrate specificity (Pinna L. A., Biochim. Biophys. Acta 1054:267-284, 1990) of this enzyme can be summarized as follows: (1) Under comparable conditions Ser is favored over Thr; (2) an acidic residue (either Asp or Glu) must be present three residues from the C-terminal end of the phosphate acceptor site; (3) additional acidic residues in positions +1, +2, +4, and +5 increase the phosphorylation rate (most physiological substrates have at least one acidic residue in these positions); (4) Asp is preferred to Glu as the provider of acidic determinants; and (5) a basic residue at the N-terminus of the acceptor site decreases the phosphorylation rate, while an acidic one will increase it. The consensus pattern is: [ST]-x(2)-[DE] where S or T is the phosphorylation site (note: this pattern is found in most of the known physiological substrates).

If phosphorylation of a specific site by any kinase is found to increase the catalytic activity or stability of the encoded desaturase protein, the phosphorylated serine or threonine residue is changed to encode a negatively charged amino acid (aspartic acid or glutamic acid) in order to permanently optimize the activity or the protein. If phosphorylation of a specific residue is found to decrease the activity or stability of the encoded desaturase, the affected serine or threonine encoding codon is altered to substitute a neutral or a positively charged amino acid that will permanently optimize the activity or stability of the protein.

EXAMPLE 4 Further Modifications and Improvements of the Saccharomyces cerevisiae OLE1 Gene for Plant Expression Using Elements Derived from Native Plant Desaturase Genes

The activity of the native or modified forms of the Saccharoznyces cerevisiae OLE1 Δ-9 desaturase gene in plant tissues may be further improved by the substitution or inclusion of elements derived from native plant desaturase genes. Favorable plant gene elements may include sequences that improve the expression of the modified gene at one or more levels, including the following: 1) transcription, 2) pre-mRNA processing, 3) mRNA transport from the nucleus to the cytoplasm, 4) mRNA stability 5) translation, 6) targeting or retention of the protein at the appropriate membrane surface or organelle surface, 7) protein folding and maturation, and 8) stability of the functional desaturase protein.

The inventors have shown that the OLE1 gene can tolerate significant modifications without losing its biological activity. These modifications include deletion of the “coiled coil” region, the addition of 239 amino acids to the N-terminus of OLE1p and truncation of 55 and 60 amino acids from the N-terminal end of the protein. The inventors have also shown that modifications of the 5′ and 3′ untranslated regions of the OLE1 mRNA can significantly affect its stability. For example, removing a short open reading frame near the 5′ “cap” region of the OLE1 mRNA increases its half-life in Saccharomyces from 12 minutes to approximately 25 minutes. The existence of elements in the mRNA that affect its stability indicate that other elements might also exist that affect the stability of an mRNA generated by a synthetic gene in another host organism.

Plant desaturase gene elements that enhance the function of the modified Δ-9 desaturase gene are identified by a 2-step method. STEP 1 involves isolating a series of DNA sequences from a cDNA that encodes a plant ER lipid biosynthetic enzyme. Those elements are linked, or inserted into regions of a native or “optimized” gene under control of a yeast promoter in a vector suitable for expression in Saccharomyces cerevisiae. The resulting vectors are then tested for their ability to produce functional desaturase enzymes in strains of Saccharomyces that contain an inactive form of the Δ-9 fatty acid desaturase gene.

In STEP 2, plant desaturase sequences from the above vectors that are found to produce a functional Δ-9 desaturase gene are used to a isolate homologous sequences from plant genomic DNA. The isolated genomic sequences are used to construct a synthetic gene that produces an mRNA that encodes the same functional desaturase protein produced by the vector in step 1. In this instance, the genomic sequences encompass the same protein coding elements as those encoded by the homologous cDNA sequence and also include genomic elements that encode the 5′ and/or 3′ untranslated regions of the plant desaturase mRNA. These combined genomic elements should differ from the cDNA derived sequences used in STEP 1 by containing authentic plant introns, (which may facilitate efficient and correct splicing of the chimeric mRNA in the plant nucleus) and signals that affect the mRNA stability, mRNA transport, and efficient translation of the mRNA in plant tissues. The chimeric plant/synthetic gene containing the genomic sequences is inserted into vectors under the control of plant seed-specific promoters and tested for expression and desaturase function in plants, including Brassica, Arabidopsis, maize and soybeans.

The following specific examples further illustrate these methods employing the Arabidopsis FAD2 gene, which encodes an ER Δ12-desaturase, as a source of plant desaturase DNA sequences. In the preferred embodiment, the source of the plant desaturase DNA would be the FAD2 homolog, or a related ER lipid biosynthetic gene, that is derived from the same plant species that is intended to be modified by the resulting vector for commercial use.

A. Substitution of the N-terminal OLE1 protein coding sequences and with N-terminal sequences from the derived from the Arabidopsis FAD2 gene.

1) A cDNA containing the FAD2, Δ-12 desaturase, mRNA coding sequence is isolated by reverse transcriptase—polymerase chain reaction (RT-PCR) of isolated mRNAs derived from Arabidopsis tissue or by direct DNA synthesis using the protein and DNA sequences set forth in SEQ ID NO:4 and SEQ ID NO:5 (open reading frame starts at +93).

2) The inventors have shown that substitution of transmembrane sequences of the OLE1 gene with transmembrane sequences from the Saccharomyces FAH2 gene abolishes Δ-9 desaturase activity. FAH2 encodes a sphingolipid fatty acid hydroxylase, which is an ER membrane protein. TMPredict analysis of the Arabidopsis FAD2 sequence indicates that the first transmembrane region of its encoded protein begins at residue +52 and a similar analysis of the OLE1 sequence indicates that its first transmembrane sequence begins at residue +113. Because the inclusion of potential membrane spanning elements from the plant desaturase could produce significant changes in the desaturase core enzyme structure that affect activity, only sequences encoding residues +1 to +52 of FAD2 are tested for functional linkages or substitutions in the 113 residue N-terminal region of OLE1.

A series of PCR oligonucleotide primers are synthesized that include a 5′ primer that complements sequences including +1 start codon of the FAD2 gene and 3′ primers that complement sequences ending, for example, at residues +20, +35 and +52 of the FAD2 gene. These are used to amplify a series of fragments of different lengths from the FAD2 cDNA that extend from the +1 codon through codon +52. A second PCR amplification is performed using a 5′ primer that is complementary to sequences that include the 5′ end of the FAD2 mRNA and the 3′ primer that includes codon +52. That amplification is done using Arabidopsis genomic DNA as a template. The amplified fragment from that reaction is cloned into a bacterial vector and subjected to DNA sequencing to detect the presence of introns within the genomic sequence. The cloned genomic fragment is also used to construct vectors for plant expression as indicated in STEP 2 of the method.

The amplified cDNA fragments is inserted into yeast expression vectors that contain the native OLE1 mRNA coding sequence under the control of the Saccharomyces galactose inducible, GAL1 promoter. Insertion of the plant DNA fragments can be done in several ways: 1) A fragment is inserted upstream of the OLE1 protein coding sequences so that its protein coding element is fused in frame to the +1 codon of the OLE1 encoded protein, 2) the codons on the plant fragment could replace the equivalent OLE2 residues starting from the +1 ATG codon (e.g. a plant DNA fragment containing codons +1→+52 replaces OLE1 codons +1→+52) and 3) the full length fragment containing codons +1→+52 of the plant gene is fused in frame to codon +114 of the OLE1 gene, replacing the OLE1 residues +1→+113 with plant desaturase residues +1→+52.

The resulting plasmids are transformed into a haploid ole1Δ::LEU2 strain of Saccharomyces. That strain contains a null, disrupted form of the OLE1 gene and therefore has a growth requirement for unsaturated fatty acids. The transformed Saccharomyces strains are grown on fatty acid depleted galactose medium to test for the ability of the induced chimeric gene to support growth of the strain without fatty acids. Transformed strains that grow on the fatty acid deficient medium are further analyzed to assess the effects of the plant sequences on desaturase function. This is done by Western blot analysis, to measure levels of the resulting desaturase protein and by fatty acid analysis of total cellular lipids, to assess the relative activity of the desaturase enzyme by comparison of the ratio of saturated to unsaturated fatty acids.

3) Using information derived from the above tests, a chimeric desaturase gene is constructed using the amplified genomic DNA from the FAD2 gene. Construction, testing, and analysis these vectors is guided by the principle that the most desirable vector is one that maximizes the use of the plant gene sequences and minimizes the use of the Saccharomyces Δ-9 desaturase gene sequences while retaining optimal desaturase function. Plant DNA fragments derived from the genomic DNA amplification that extend from the 5′ end of the mRNA sequence to the longest sequence that produces optimal desaturase function in yeast are inserted into a vector containing the native Δ-9 desaturase gene (or one of its modified forms produced by the methods described above). The fragment is inserted into the vector so that the 3′ end of its protein coding sequence produces an mRNA that generates a protein sequence identical to its counterpart derived from the FAD2 cDNA sequences. The resulting chimeric desaturase gene, which now encodes an mRNA that includes the FAD2 5′ untranslated region in addition to the modified protein coding sequences, is placed into a plant expression vector under the control of a suitable plant promoter and plant termination/polyadenylation sequences.

4) The resulting vectors containing the plant/yeast chimeric desaturase sequences are transformed into plants for testing and analysis of desaturase function. Suitable test plants include Arabidopsis thaliana, and Brassica napis. A method for transformation and analysis of desaturase gene expression in Arabidopsis is provided above. A method for transformation and analysis of yeast desaturase expression in Brassica napis is described in U.S. Pat. No. 5,777,201 to Poutre et al. (incorporated by reference herein).

B. Insertion or substitution of Arabidopsis FAD2 C-terminal protein coding sequences and 3′ mRNA untranslated region sequences into native and modified forms of the OLE1 gene.

The inventors have previously shown that proteins encoded by the Saccharomyces ELO2 and ELO3 genes contain a series of charged residues in their C-terminal region. These proteins are located on the ER surface and function in the biosynthesis of very long chain fatty acids as described in Oh et al. (J. Biol. Chem. 272: 17376-17384, 1997) (incorporated by reference herein). They further showed that deletion of the region containing the charged residues causes the proteins to be mislocalized from their normal cellular locations in the endoplasmic reticulum, resulting in reduced function. Similar clusters of charged residues occurs in the C-terminal region of the OLE1 gene that are apparently associated with ER retention or localization. These residues do not appear to be a part of the functional cytochrome b₅ domain. A detailed comparison of the C-terminal OLE1 and the Arabidopsis FAD2 sequences show that the plant desaturase has similar, but not identical, clusters of charged residues to those in the OLE1 gene. These sequences are shown below:

SEQ ID Nos: 6 and 7:

Comparison of the charged carboxyl terminal amino acids of Ole1p (SEQ ID NO:7) and the Arabidopsis Fad2p desaturase (SEQ ID NO:6) (The region of the OLE1 gene shown does not appear to be a functional part of its cytochrome b₅ domain).

      +− + −    − −+−   −++        + A. thaliana WYVAMYREAK ECIYVEPDRE GDKKGVYWYN NKL* FAD2       +− +     +   ++   −  −  + S. cerevisiae MRVAVIKESK NSAIRMASKR GEIYETGKFF * OLE1

Methods similar to those shown in Section A can be used to identify Arabidopsis FAD2 sequences that can replace the OLE1 C-terminal sequences to optimize gene expression, membrane targeting and ER retention of the chimeric enzyme.

1) A series of oligonucleotide primers for PCR amplification are synthesized for isolation of elements in the C-terminal region of the FAD2 gene. A FAD2 DNA fragment encompassing that region is generated by PCR amplification of the cDNA clone. Alternatively, given the smaller size of the fragment it or modified forms of the plant fragment may be generated directly by DNA synthesis. A fragment containing that region and its flanking 3′ untranslated region also is generated by PCR amplification of Arabidopsis genomic DNA as described above. That fragment is cloned into an appropriate vector and sequenced. as also described.

2) Vectors are constructed that contain the plant DNA fragments linked to or substituted into the OLE1 C-terminal coding region as described in Section A. In this instance, the plant DNA fragments are linked in frame to the carboxyl terminal residues of the OLE1 protein coding region.

3) The resulting vectors are transformed into the Saccharomyces ole1Δ strain and tested for desaturase function as described in Section A.

4) Using information derived from the above tests, chimeric desaturase genes containing the C-terminal plant sequences that produce functional desaturases are constructed using the amplified genomic DNA from the FAD2 gene, according to the principles outlined in Section A. The resulting sequences are employed to construct vectors that will express the chimeric plant/yeast gene under control of plant promoter and plant termination/polyadenylation sequences. Those vectors are transformed into plants for testing and analysis of desaturase function as described above.

The present invention is not limited to the embodiments described and exemplified above, but is capable of variation and modification without departure from the scope of the appended claims.

8 1 1555 DNA Saccharomyces cerevisiae 1 tacaacaaag atgccaactt ctggaactac tattgaattg attgacgacc aatttccaaa 60 ggatgactct gccagcagtg gcattgtcga cgaagtcgac ttaacggaag ctaatatttt 120 ggctactggt ttgaataaga aagcaccaag aattgtcaac ggttttggtt ctttaatggg 180 ctccaaggaa atggtttccg tggaattcga caagaaggga aacgaaaaga agtccaattt 240 ggatcgtctg ctagaaaagg acaaccaaga aaaagaagaa gctaaaacta aaattcacat 300 ctccgaacaa ccatggactt tgaataactg gcaccaacat ttgaactggt tgaacatggt 360 tcttgtttgt ggtatgccaa tgattggttg gtacttcgct ctctctggta aagtaccttt 420 gcatttaaac gttttccttt tctccgtttt ctactacgct gtcggtggtg tttctattac 480 tgccggttac catagattat ggtctcacag atcttactcc gctcactggc cattgagatt 540 attctacgct atcttcggtt gtgcttccgt tgaagggtcc gctaaatggt ggggccactc 600 tcacagaatt caccatcgtt acactgatac cttgagagat ccttatgacg ctcgtagagg 660 tctatggtac tcccacatgg gatggatgct tttgaagcca aatccaaaat acaaggctag 720 agctgatatt accgatatga ctgatgattg gaccattaga ttccaacaca gacactacat 780 cttgttgatg ttattaaccg ctttcgtcat tccaactctt atctgtggtt actttttcaa 840 cgactatatg ggtggtttga tctatgccgg ttttattcgt gtctttgtca ttcaacaagc 900 taccttttgc attaactcca tggctcatta catcggtacc caaccattcg atgacagaag 960 aacccctcgt gacaactgga ttactgccat tgttactttc ggtgaaggtt accataactt 1020 ccaccacgaa ttcccaactg attacagaaa cgctattaag tggtaccaat acgacccaac 1080 taaggttatc atctatttga cttctttagt tggtctagca tacgacttga agaaattctc 1140 tcaaaatgct attgaagaag ccttgattca acaagaacaa aagaagatca ataaaaagaa 1200 ggctaagatt aactggggtc cagttttgac tgatttgcca atgtgggaca aacaaacctt 1260 cttggctaag tctaaggaaa acaagggttt ggttatcatt tctggtattg ttcacgacgt 1320 atctggttat atctctgaac atccaggtgg tgaaacttta attaaaactg cattaggtaa 1380 ggacgctacc aaggctttca gtggtggtgt ctaccgtcac tcaaatgccg ctcaaaatgt 1440 cttggctgat atgagagtgg ctgttatcaa ggaaagtaag aactctgcta ttagaatggc 1500 tagtaagaga ggtgaaatct acgaaactgg taagttcttt taagcatcac attac 1555 2 510 PRT Saccharomyces cerevisiae 2 Met Pro Thr Ser Gly Thr Thr Ile Glu Leu Ile Asp Asp Gln Phe Pro 1 5 10 15 Lys Asp Asp Ser Ala Ser Ser Gly Ile Val Asp Glu Val Asp Leu Thr 20 25 30 Glu Ala Asn Ile Leu Ala Thr Gly Leu Asn Lys Lys Ala Pro Arg Ile 35 40 45 Val Asn Gly Phe Gly Ser Leu Met Gly Ser Lys Glu Met Val Ser Val 50 55 60 Glu Phe Asp Lys Lys Gly Asn Glu Lys Lys Ser Asn Leu Asp Arg Leu 65 70 75 80 Leu Glu Lys Asp Asn Gln Glu Lys Glu Glu Ala Lys Thr Lys Ile His 85 90 95 Ile Ser Glu Gln Pro Trp Thr Leu Asn Asn Trp His Gln His Leu Asn 100 105 110 Trp Leu Asn Met Val Leu Val Cys Gly Met Pro Met Ile Gly Trp Tyr 115 120 125 Phe Ala Leu Ser Gly Lys Val Pro Leu His Leu Asn Val Phe Leu Phe 130 135 140 Ser Val Phe Tyr Tyr Ala Val Gly Gly Val Ser Ile Thr Ala Gly Tyr 145 150 155 160 His Arg Leu Trp Ser His Arg Ser Tyr Ser Ala His Trp Pro Leu Arg 165 170 175 Leu Phe Tyr Ala Ile Phe Gly Cys Ala Ser Val Glu Gly Ser Ala Lys 180 185 190 Trp Trp Gly His Ser His Arg Ile His His Arg Tyr Thr Asp Thr Leu 195 200 205 Arg Asp Pro Tyr Asp Ala Arg Arg Gly Leu Trp Tyr Ser His Met Gly 210 215 220 Trp Met Leu Leu Lys Pro Asn Pro Lys Tyr Lys Ala Arg Ala Asp Ile 225 230 235 240 Thr Asp Met Thr Asp Asp Trp Thr Ile Arg Phe Gln His Arg His Tyr 245 250 255 Ile Leu Leu Met Leu Leu Thr Ala Phe Val Ile Pro Thr Leu Ile Cys 260 265 270 Gly Tyr Phe Phe Asn Asp Tyr Met Gly Gly Leu Ile Tyr Ala Gly Phe 275 280 285 Ile Arg Val Phe Val Ile Gln Gln Ala Thr Phe Cys Ile Asn Ser Leu 290 295 300 Ala His Tyr Ile Gly Thr Gln Pro Phe Asp Asp Arg Arg Thr Pro Arg 305 310 315 320 Asp Asn Trp Ile Thr Ala Ile Val Thr Phe Gly Glu Gly Tyr His Asn 325 330 335 Phe His His Glu Phe Pro Thr Asp Tyr Arg Asn Ala Ile Lys Trp Tyr 340 345 350 Gln Tyr Asp Pro Thr Lys Val Ile Ile Tyr Leu Thr Ser Leu Val Gly 355 360 365 Leu Ala Tyr Asp Leu Lys Lys Phe Ser Gln Asn Ala Ile Glu Glu Ala 370 375 380 Leu Ile Gln Gln Glu Gln Lys Lys Ile Asn Lys Lys Lys Ala Lys Ile 385 390 395 400 Asn Trp Gly Pro Val Leu Thr Asp Leu Pro Met Trp Asp Lys Gln Thr 405 410 415 Phe Leu Ala Lys Ser Lys Glu Asn Lys Gly Leu Val Ile Ile Ser Gly 420 425 430 Ile Val His Asp Val Ser Gly Tyr Ile Ser Glu His Pro Gly Gly Glu 435 440 445 Thr Leu Ile Lys Thr Ala Leu Gly Lys Asp Ala Thr Lys Ala Phe Ser 450 455 460 Gly Gly Val Tyr Arg His Ser Asn Ala Ala Gln Asn Val Leu Ala Asp 465 470 475 480 Met Arg Val Ala Val Ile Lys Glu Ser Lys Asn Ser Ala Ile Arg Met 485 490 495 Ala Ser Lys Arg Gly Glu Ile Tyr Glu Thr Gly Lys Phe Phe 500 505 510 3 1555 DNA Artificial Sequence synthetic yeast delta-9 desaturase gene modified for expression in plants 3 ggatccaaca atgcctactt ctggaactac tatcgagctt atcgatgatc aattccctaa 60 ggatgattct gcttcttctg gaatcgttga tgaggttgat cttactgagg ctaacatcct 120 tgctactgga cttaacaaga aggctcctag aatcgttaac ggattcggat ctcttatggg 180 atctaaggag atggtttctg ttgagttcga taagaaggga aacgagaaga agtctaacct 240 tgatagactt cttgagaagg ataaccaaga gaaggaggag gctaagacta agatccatat 300 ctctgagcaa ccttggactc tcaacaactg gcatcaacat ctcaactggc tcaacatggt 360 gctcgtctgt ggaatgccta tgatcggatg gtacttcgct ctctctggaa aagtgcctct 420 ccatctcaac gttttcctct tctctgtctt ctactacgct gttggaggag tgtctatcac 480 tgctggatac catagactct ggtctcatag atcttactct gctcattggc ctcttagact 540 cttctacgct atctttggat gtgcttctgt tgagggatct gctaagtggt ggggacattc 600 tcatagaatc catcatagat acactgatac tcttagagat ccttacgatg ctagaagagg 660 actttggtac tctcatatgg gatggatgct tcttaagcct aaccctaagt acaaggctag 720 agctgatatc actgatatga ctgatgattg gactatcaga ttccaacata gacattacat 780 cttgctcatg ctccttactg ctttcgtgat ccctactctc atctgtggat acttcttcaa 840 cgattacatg ggaggactca tctacgctgg attcatcaga gtgttcgtca tccaacaagc 900 tactttctgt atcaactcta tggctcatta catcggaact caacctttcg atgatagaag 960 aactcctaga gataactgga tcactgctat cgttactttc ggagagggat accataactt 1020 ccatcatgag ttccctactg attatagaaa cgctatcaag tggtaccaat acgatcctac 1080 taaagtgatc atctacttga cttctctcgt gggacttgct tacgatctca agaagttctc 1140 tcaaaacgct atcgaggagg ctcttatcca acaagagcaa aagaagatca acaagaagaa 1200 ggctaagatt aattggggac ctgttcttac tgatcttcct atgtgggata agcaaacttt 1260 ccttgctaag tctaaggaga acaagggact tgttatcatc tctggaatcg ttcatgatgt 1320 ttctggatac atctctgagc atcctggagg agagacttta attaagactg ctcttggaaa 1380 ggatgctact aaggctttct ctggaggagt ttacagacat tctaacgctg ctcaaaacgt 1440 gcttgctgat atgagagttg ctgttatcaa ggagtctaag aactctgcta tcagaatggc 1500 ttctaagaga ggagagatct acgagactgg aaagttcttc tgatctagag gatcc 1555 4 383 PRT Arabidopsis thaliana 4 Met Gly Ala Gly Gly Arg Met Pro Val Pro Thr Ser Ser Lys Lys Ser 1 5 10 15 Glu Thr Asp Thr Thr Lys Arg Val Pro Cys Glu Lys Pro Pro Phe Ser 20 25 30 Val Gly Asp Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 35 40 45 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Ser Asp Ile Ile Ile Ala Ser 50 55 60 Cys Phe Tyr Tyr Val Ala Thr Asn Tyr Phe Ser Leu Leu Pro Gln Pro 65 70 75 80 Leu Ser Tyr Leu Ala Trp Pro Leu Tyr Trp Ala Cys Gln Gly Cys Val 85 90 95 Leu Thr Gly Ile Trp Val Ile Ala His Glu Cys Gly His His Ala Phe 100 105 110 Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser 115 120 125 Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg His 130 135 140 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys 145 150 155 160 Gln Lys Ser Ala Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 Gly Arg Ile Met Met Leu Thr Val Gln Phe Val Leu Gly Trp Pro Leu 180 185 190 Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Phe Ala Cys 195 200 205 His Phe Phe Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu Gln 210 215 220 Ile Tyr Leu Ser Asp Ala Gly Ile Leu Ala Val Cys Phe Gly Leu Tyr 225 230 235 240 Arg Tyr Ala Ala Ala Gln Gly Met Ala Ser Met Ile Cys Leu Tyr Gly 245 250 255 Val Pro Leu Leu Ile Val Asn Ala Phe Leu Val Leu Ile Thr Tyr Leu 260 265 270 Gln His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp Asp 275 280 285 Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile Leu 290 295 300 Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His Leu 305 310 315 320 Phe Ser Thr Met Pro His Tyr Asn Ala Met Glu Ala Thr Lys Ala Ile 325 330 335 Lys Pro Ile Leu Gly Asp Tyr Tyr Gln Phe Asp Gly Thr Pro Trp Tyr 340 345 350 Val Ala Met Tyr Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro Asp 355 360 365 Arg Glu Gly Asp Lys Lys Gly Val Tyr Trp Tyr Asn Asn Lys Leu 370 375 380 5 1372 DNA Arabidopsis thaliana 5 agagagagag attctgcgga ggagcttctt cttcgtaggg tgttcatcgt tattaacgtt 60 atcgccccta cgtcagctcc atctccagaa acatgggtgc aggtggaaga atgccggttc 120 ctacttcttc caagaaatcg gaaaccgaca ccacaaagcg tgtgccgtgc gagaaaccgc 180 ctttctcggt gggagatctg aagaaagcaa tcccgccgca ttgtttcaaa cgctcaatcc 240 ctcgctcttt ctcctacctt atcagtgaca tcattatagc ctcatgcttc tactacgtcg 300 ccaccaatta cttctctctc ctccctcagc ctctctctta cttggcttgg ccactctatt 360 gggcctgtca aggctgtgtc ctaactggta tctgggtcat agcccacgaa tgcggtcacc 420 acgcattcag cgactaccaa tggctggatg acacagttgg tcttatcttc cattccttcc 480 tcctcgtccc ttacttctcc tggaagtata gtcatcgccg tcaccattcc aacactggat 540 ccctcgaaag agatgaagta tttgtcccaa agcagaaatc agcaatcaag tggtacggga 600 aatacctcaa caaccctctt ggacgcatca tgatgttaac cgtccagttt gtcctcgggt 660 ggcccttgta cttagccttt aacgtctctg gcagaccgta tgacgggttc gcttgccatt 720 tcttccccaa cgctcccatc tacaatgacc gagaacgcct ccagatatac ctctctgatg 780 cgggtattct agccgtctgt tttggtcttt accgttacgc tgctgcacaa gggatggcct 840 cgatgatctg cctctacgga gtaccgcttc tgatagtgaa tgcgttcctc gtcttgatca 900 cttacttgca gcacactcat ccctcgttgc ctcactacga ttcatcagag tgggactggc 960 tcaggggagc tttggctacc gtagacagag actacggaat cttgaacaag gtgttccaca 1020 acattacaga cacacacgtg gctcatcacc tgttctcgac aatgccgcat tataacgcaa 1080 tggaagctac aaaggcgata aagccaattc tgggagacta ttaccagttc gatggaacac 1140 cgtggtatgt agcgatgtat agggaggcaa aggagtgtat ctatgtagaa ccggacaggg 1200 aaggtgacaa gaaaggtgtg tactggtaca acaataagtt atgagcatga tggtgaagaa 1260 attgtcgacc tttctcttgt ctgtttgtct tttgttaaag aagctatgct tcgttttaat 1320 aatcttattg tccattttgt tgtgttatga cattttggct gctcattatg tt 1372 6 6 000 7 33 PRT Arabidopsis thaliana 7 Trp Tyr Val Ala Met Tyr Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu 1 5 10 15 Pro Asp Arg Glu Gly Asp Lys Lys Gly Val Tyr Trp Tyr Asn Asn Lys 20 25 30 Leu 8 30 PRT Saccharomyces cerevisiae 8 Met Arg Val Ala Val Ile Lys Glu Ser Lys Asn Ser Ala Ile Arg Met 1 5 10 15 Ala Ser Lys Arg Gly Glu Ile Tyr Glu Thr Gly Lys Phe Phe 20 25 30 

We claim:
 1. A synthetic fatty acid desaturase gene for expression in a multicellular plant, the gene comprising SEQ ID NO:3, wherein the gene is customized from a naturally occurring cytosolic Δ-9 desaturase gene from Saccharomyces cerevisiae for expression in a plant cytoplasm.
 2. The synthetic gene of claim 1, which further comprises an expression regulatory sequence from a plant gene encoding an ER biosynthetic pathway enzyme.
 3. The synthetic gene of claim 1, customized for expression in a monocotyledonous plant.
 4. The synthetic gene of claim 1, customized for expression in a dicotyledonous plant.
 5. The synthetic gene of claim 1, customized for expression in a plant genus selected from the group consisting of Arabidopsis, Brassica, Phaeseolus, Oryza, Olea, Elaeis (Oil Palm) and Zea.
 6. The synthetic gene of claim 1, customized from a naturally occurring gene comprising both a desaturase domain and a cyt b₅ domain.
 7. The synthetic gene of claim 1, wherein the gene is a chimeric gene comprising a desaturase domain and a heterologous cyt b₅ domain.
 8. The synthetic gene of claim 1, customized from a naturally occurring gene such that the synthetic gene and the naturally occurring gene encode an identical amino acid sequence.
 9. The synthetic gene of claim 8, wherein the synthetic gene and the naturally occurring gene encode SEQ ID NO:2.
 10. The synthetic gene of claim 1, customized from a naturally occurring gene such that the synthetic gene and the naturally occurring gene encode a similar amino acid sequence.
 11. The synthetic gene of claim 1, customized from a naturally occurring gene such that the synthetic gene and the naturally occurring gene encode a similar amino acid sequence, and the synthetic gene possesses improved stability or catalytic activity as compared with the naturally occurring gene. 